Anti-sialyl tn chimeric antigen receptors

ABSTRACT

The invention provides improved compositions for adoptive cell therapies for cancers that express the glycoepitope STn on TAG-72.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/317,950, filed Apr. 4, 2016, and claimsthe benefit under 35 U.S.C. § 119(a)-(d) of Korean Patent ApplicationNo. 10-2015-0122727, filed Aug. 31, 2015, each of which is incorporatedby reference herein in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is BLBD_066_02WO_ST25.txt. The text file is 38 KB,was created on Aug. 30, 2016, and is being submitted electronically viaEFS-Web, concurrent with the filing of the specification.

BACKGROUND Technical Field

The present invention relates to improved compositions and methods fortreating cancer. More particularly, the invention relates to improvedchimeric antigen receptors (CARs) comprising anti-STn antibodies orantigen binding fragments thereof, immune effector cells geneticallymodified to express these CARs, and use of these compositions toeffectively treat STn expressing cancers.

Description of the Related Art

Sialyl-Tn antigen (STn) is a short O-glycan containing a sialic acidresidue α2,6-linked to GalNAcα-O-Ser/Thr. The biosynthesis of STn ismediated by a specific sialyltransferase termed ST6GalNAc I, whichcompetes with O-glycans elongating glycosyltransferases and preventscancer cells from exhibiting longer O-glycans. Various epitopes of theSTn antigen are expressed on glycoproteins including, mucins andmucin-like proteins such as mucin 1 (MUC1), mucin 16 (MUC16), and tumorassociated glycoprotein 72 (TAG-72).

STn is weakly expressed by fetal and normal adult tissues, but is alsoexpressed by more than 80% of human carcinomas. STn detection isgenerally associated with adverse outcome and decreased overall survivalfor the cancer patients. Because of its pan-carcinoma expressionassociated with an adverse outcome, an anti-cancer vaccine, namedTheratope, has been designed towards the STn antigen. In spite of thegreat enthusiasm around this immunotherapy, Theratope failed on PhaseIII clinical trial.

Another failed clinical trial for treating STn expressing cancers usedT-cells engineered with chimeric antigen receptors (CAR). Scientists atCell Genesys used anti-STn CAR T cells that bind the STn antigenexpressed on the glycoprotein TAG-72 in patients with metastatic coloncancer. Patients were given intravenous or intrahepatic infusions ofanti-STn CAR T cells. The anti-STn CARS did not induce any antitumoractivity.

Accordingly, the potential of efficient immunotherapy strategiestargeting STn has yet to be realized.

BRIEF SUMMARY

The invention generally provides improved vectors for generating T celltherapies and methods of using the same. More particularly, theinvention provides anti-sialyl Tn antigen (sTn) CAR molecules and theiruse in treating, preventing, or ameliorating cancers that express sTnexpressing glycoproteins.

In various embodiments, a chimeric antigen receptor (CAR) is providedcomprising: an extracellular domain that comprises: a) an anti-STnantibody or antigen binding fragment thereof that binds one or moreepitopes of an STn antigen or an STn antigen expressed on aglycoprotein, wherein the anti-STn antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising CDRL1-CDRL3sequences set forth in SEQ ID NOs: 1-3, 9-11, or 17-19, and a variableheavy chain sequence comprising CDRH1-CDRH3 sequences set forth in SEQID NOs: 4-6, 12-14, or 20-22; b) a transmembrane domain; c) one or moreintracellular co-stimulatory signaling domains; and d) a primarysignaling domain.

In various embodiments, the STn antigen is expressed on a glycoproteinselected from the group consisting of mucins or mucin-likeglycoproteins.

In particular embodiments, the STn antigen is expressed on a mucinselected from the group consisting of: mucin 1 and mucin 16.

In certain embodiments, the STn antigen is expressed on a mucin-likeprotein.

In various embodiments, the mucin-like protein is TAG-72.

In particular embodiments, the anti-STn antibody or antigen bindingfragment that binds the STn antigen is selected from the groupconsisting of: a Camel Ig, Ig NAR, Fab fragments, Fab′ fragments,F(ab)′2 fragments, F(ab)′3 fragments, Fv, single chain Fv antibody(“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody,disulfide stabilized Fv protein (“dsFv”), and single-domain antibody(sdAb, nanobody).

In certain embodiments, the anti-STn antibody or antigen bindingfragment that binds the STn antigen is an scFv.

In particular embodiments, the anti-STn antibody or antigen bindingfragment thereof comprises one or more light chain CDRs as set forth inany one of SEQ ID NOs: 1-3 and/or one or more heavy chain CDRs as setforth in any one of SEQ ID NOs: 4-6.

In some embodiments, the anti-STn antibody or antigen binding fragmentthereof comprises one or more light chain CDRs as set forth in any oneof SEQ ID NOs: 9-11 and/or one or more heavy chain CDRs as set forth inany one of SEQ ID NOs: 12-14.

In additional embodiments, the anti-STn antibody or antigen bindingfragment thereof comprises one or more light chain CDRs as set forth inany one of SEQ ID NOs: 17-19 and/or one or more heavy chain CDRs as setforth in any one of SEQ ID NOs: 20-22.

In some embodiments, the anti-STn antibody or antigen binding fragmentthereof comprises a variable light chain sequence as set forth in anyone of SEQ ID NOs: 7, 15, or 23, and/or a variable heavy chain sequenceas set forth in any one of SEQ ID NOs: 8, 16, or 24.

In certain embodiments, the anti-STn antibody or antigen bindingfragment thereof comprises a variable light chain sequence as set forthin SEQ ID NO: 7 and/or a variable heavy chain sequence as set forth inSEQ ID NO: 8.

In further embodiments, the anti-STn antibody or antigen bindingfragment thereof comprises a variable light chain sequence as set forthin SEQ ID NO: 15 and/or a variable heavy chain sequence as set forth inSEQ ID NO: 16.

In particular embodiments, the anti-STn antibody or antigen bindingfragment thereof comprises a variable light chain sequence as set forthin SEQ ID NO: 23 and/or a variable heavy chain sequence as set forth inSEQ ID NO: 24.

In further embodiments, the transmembrane domain is from a polypeptideselected from the group consisting of: alpha or beta chain of the T-cellreceptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27,CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154,and PD1.

In additional embodiments, the transmembrane domain is from apolypeptide selected from the group consisting of: CD8α; CD4, CD45, PD1,and CD152.

In some embodiments, the transmembrane domain is from CD8α.

In further embodiments, the transmembrane domain is from PD1.

In particular embodiments, the transmembrane domain is from CD152.

In further embodiments, the one or more co-stimulatory signaling domainsare from a co-stimulatory molecule selected from the group consistingof: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11,CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137(4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

In certain embodiments, the one or more co-stimulatory signaling domainsare from a co-stimulatory molecule selected from the group consistingof: CD28, CD134, and CD137.

In some embodiments, the one or more co-stimulatory signaling domains isfrom CD28.

In some embodiments, the one or more co-stimulatory signaling domains isfrom CD134.

In some embodiments, the one or more co-stimulatory signaling domains isfrom CD137.

In particular embodiments, the primary signaling domain is isolated froma polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ,CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

In particular embodiments, the primary signaling domain is isolated fromCD3ζ.

In additional embodiments, the CAR further comprises a hinge regionpolypeptide.

In certain embodiments, the hinge region polypeptide comprises a hingeregion of CD8α.

In further embodiments, the hinge region polypeptide comprises a hingeregion of PD1.

In particular embodiments, the hinge region polypeptide comprises ahinge region of CD152.

In additional embodiments, the CAR further comprises a spacer region.

In further embodiments, the spacer region polypeptide comprises CH2 andCH3 regions of IgG1, IgG4, or IgD.

In further embodiments, the CAR further comprises a signal peptide.

In particular embodiments, the signal peptide comprises an IgG1 heavychain signal polypeptide, a CD8a signal polypeptide, or a human GM-CSFreceptor alpha signal polypeptide.

In particular embodiments, a CAR comprises an amino acid sequence setforth in any one of SEQ ID NOs: 25-27.

In particular embodiments, a CAR comprises an amino acid sequence setforth in SEQ ID NO: 25.

In particular embodiments, a CAR comprises an amino acid sequence setforth in SEQ ID NO: 26.

In particular embodiments, a CAR comprises an amino acid sequence setforth in SEQ ID NO: 27.

In various embodiments, a polypeptide comprising the amino acid sequenceof the CAR contemplated herein is provided.

In various embodiments, a polynucleotide encoding a CAR contemplatedherein is provided.

In various embodiments, a vector comprising a polynucleotide encoding aCAR contemplated herein is provided.

In certain embodiments, the vector is an expression vector.

In particular embodiments, the vector is an episomal vector.

In further embodiments, the vector is a viral vector.

In further embodiments, the vector is a retroviral vector.

In particular embodiments, the vector is a lentiviral vector.

In further embodiments, the lentiviral vector is selected from the groupconsisting essentially of: human immunodeficiency virus 1 (HIV-1); humanimmunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus; caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (FIV; bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV).

In particular embodiments, the vector comprises a left (5′) retroviralLTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA flap(cPPT/FLAP), a retroviral export element; a promoter operably linked tothe polynucleotide; and a right (3′) retroviral LTR.

In further embodiments, the vector further comprises a heterologouspolyadenylation sequence.

In particular embodiments, the vector further comprises a hepatitis Bvirus posttranscriptional regulatory element (HPRE) or woodchuckpost-transcriptional regulatory element (WPRE).

In additional embodiments, the promoter of the 5′ LTR is replaced with aheterologous promoter.

In further embodiments, the heterologous promoter is a cytomegalovirus(CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or a Simian Virus40 (SV40) promoter.

In some embodiments, the 5′ LTR or 3′ LTR is a lentivirus LTR.

In certain embodiments, the 3′ LTR comprises one or more modifications.

In certain embodiments, the 3′ LTR comprises one or more deletions.

In particular embodiments, the 3′ LTR is a self-inactivating (SIN) LTR.

In particular embodiments, the polyadenylation sequence is a bovinegrowth hormone polyadenylation or signal rabbit β-globin polyadenylationsequence.

In additional embodiments, the polynucleotide comprises an optimizedKozak sequence.

In additional embodiments, the promoter operably linked to thepolynucleotide is selected from the group consisting of: acytomegalovirus immediate early gene promoter (CMV), an elongationfactor 1 alpha promoter (EF1-α), a phosphoglycerate kinase-1 promoter(PGK), a ubiquitin-C promoter (UBQ-C), a cytomegalovirusenhancer/chicken beta-actin promoter (CAG), polyoma enhancer/herpessimplex thymidine kinase promoter (MC1), a beta actin promoter (β-ACT),a simian virus 40 promoter (SV40), and a myeloproliferative sarcomavirus enhancer, negative control region deleted, d1587rev primer-bindingsite substituted (MND) promoter.

In various embodiments, an immune effector cell comprising a vectorencoding a CAR contemplated herein is provided.

In particular embodiments, the immune effector cell is selected from thegroup consisting of: a T lymphocyte, a natural killer T (NKT) cell and anatural killer (NK) cell.

In some embodiments, the immune effector cell is transduced with avector contemplated herein and is activated and stimulated in thepresence of an inhibitor of the PI3K pathway, thereby maintainingproliferation of the transduced immune effector cells compared to theproliferation of transduced immune effector cells that were activatedand stimulated in the absence of the inhibitor of the PI3K pathway.

In particular embodiments, the immune effector cell activated andstimulated in the presence of the inhibitor of PI3K pathway hasincreased expression of i) one or more markers selected from the groupconsisting of: CD62L, CD127, CD197, and CD38 or ii) all of the markersCD62L, CD127, CD197, and CD38 compared to an immune effector cellactivated and stimulated in the absence of the inhibitor of PI3Kpathway.

In particular embodiments, the immune effector cell activated andstimulated in the presence of the inhibitor of PI3K pathway hasincreased expression of i) one or more markers selected from the groupconsisting of: CD62L, CD127, CD27, and CD8 or ii) all of the markersCD62L, CD127, CD27, and CD8 compared to an immune effector cellactivated and stimulated in the absence of the inhibitor of PI3Kpathway.

In one embodiment, the PI3K inhibitor is ZSTK474.

In various embodiments, a composition is provided comprising the immuneeffector cell contemplated herein and a physiologically acceptableexcipient.

In various embodiments, a method of generating an immune effector cellcomprising a CAR contemplated herein is provided comprising introducinginto an immune effector cell a vector encoding a CAR contemplatedherein.

In particular embodiments, the method further comprises stimulating theimmune effector cell and inducing the cell to proliferate by contactingthe cell with antibodies that bind CD3 and antibodies that bind to CD28;thereby generating a population of immune effector cells.

In certain embodiments, the immune effector cell is stimulated andinduced to proliferate before introducing the vector.

In additional embodiments, the immune effector cells comprise Tlymphocytes.

In some embodiments, the T lymphocytes comprise natural killer T (NKT)cells.

In some embodiments, the immune effector cells comprise NK cells.

In particular embodiments, the cells are the activated and stimulated inthe presence of an inhibitor of the PI3K pathway, thereby maintainingproliferation of the transduced immune effector cells compared to theproliferation of immune effector cells that are activated and stimulatedin the absence of the inhibitor of the PI3K pathway

In some embodiments, the immune effector cells activated and stimulatedin the presence of the inhibitor of PI3K pathway have increasedexpression of i) one or more markers selected from the group consistingof: CD62L, CD127, CD197, and CD38 or ii) all of the markers CD62L,CD127, CD197, and CD38 compared to immune effector cells activated andstimulated in the absence of the inhibitor of PI3K pathway.

In particular embodiments, the immune effector cell activated andstimulated in the presence of the inhibitor of PI3K pathway hasincreased expression of i) one or more markers selected from the groupconsisting of: CD62L, CD127, CD27, and CD8 or ii) all of the markersCD62L, CD127, CD27, and CD8 compared to an immune effector cellactivated and stimulated in the absence of the inhibitor of PI3Kpathway.

In one embodiment, the PI3K inhibitor is ZSTK474.

In various embodiments, method for increasing the cytotoxicity in cancercells that express STn on glycoprotein in a subject is provided,comprising administering to the subject an amount of a compositioncontemplated herein sufficient to increase the cytotoxicity in cancercells that express STn compared to the cytotoxicity of the cancer cellsthat express STn prior to the administration.

In various embodiments, a method for decreasing the number of cancercells expressing STn on a glycoprotein in a subject is provided,comprising administering to the subject an amount of a compositioncontemplated herein sufficient to decrease the number of cancer cellsthat express STn compared to the number of the cancer cells that expressSTn prior to the administration.

In various embodiments, a method of treating a cancer in a subject inneed thereof, is provided comprising administering to the subject atherapeutically effect amount of a composition contemplated herein.

In particular embodiments, the cancer is a solid cancer.

In further embodiments, the cancer is esophageal cancer, bladder cancer,kidney cancer, lung cancer, ovarian cancer, cervical cancer, pancreaticcancer, choleangiocarcinoma, gastric cancer, colon cancer, or breastcancer.

In one embodiment, the cancer is esophageal cancer.

In one embodiment, the cancer is bladder cancer.

In one embodiment, the cancer is kidney cancer.

In one embodiment, the cancer is lung cancer.

In one embodiment, the cancer is ovarian cancer.

In one embodiment, the cancer is cervical cancer.

In one embodiment, the cancer is pancreatic cancer.

In one embodiment, the cancer is choleangiocarcinoma.

In one embodiment, the cancer is gastric cancer.

In one embodiment, the cancer is colon cancer.

In one embodiment, the cancer is breast cancer.

In particular embodiments, the cancer is a liquid cancer.

In one embodiment, the liquid cancer is a hematological malignancy.

In one embodiment, the hematological malignancy is selected from thegroup consisting of: acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma(MM), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

In one embodiment, the hematological malignancy is CLL.

In various embodiments, a method for ameliorating at least one or moresymptoms associated with a cancer expressing STn on a glycoprotein in asubject is provided, comprising administering to the subject an amountof a composition contemplated herein sufficient to ameliorate at leastone symptom associated with cancer cells that express STn.

In particular embodiments, the one or more symptoms ameliorated areselected from the group consisting of: weakness, fatigue, shortness ofbreath, easy bruising and bleeding, frequent infections, enlarged lymphnodes, distended or painful abdomen, bone or joint pain, fractures,unplanned weight loss, poor appetite, night sweats, persistent mildfever, and decreased urination.

In any of the preceding embodiments, the STn antigen is expressed on theglycoprotein, TAG-72.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a schematic of anti-STn CAR constructs.

FIG. 2 shows anti-STn CAR T cells (E) but not untransduced control Tcells kill STn expressing LS174T cells (T) in co-culture at various E:Tratios.

FIG. 3A shows the vector copy number (VCN) in T cells transduced withlentiviral vector encoding an anti-STn CAR.

FIG. 3B shows the cell surface expression of an anti-STn CAR in T cellstransduced with lentiviral vector encoding the anti-STn CAR.

FIG. 4 shows the anti-cancer activity of an exemplary anti-STn CAR. FIG.4A: Anti-STn CART cells secrete IFNγ when co-cultured with Jurkat andLS174T cell lines that express STn on TAG72. FIG. 4B: Anti-STn CAR Tcells but not untransduced T cell kill LS174T cell line in co-culture.FIG. 4C: Anti-STn CAR T cells (E) show dose-dependent cytotoxicity whenco-cultured with LS174T cells (T) at various E:T ratios.

FIG. 5 shows that administration of anti-STn CART cells delay tumoroutgrowth in NOD scid gamma (NSG) mice that received sub-cutaneous colonadenocarcinoma cells (LS174T).

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

SEQ ID NOs: 1-24 set forth amino acid sequences of exemplary light chainCDR sequences, heavy chain CDR sequences, variable domain light chains,and variable domain heavy chains for anti-STn CARs contemplated herein.

SEQ ID NOs: 25-27 set forth the amino acid sequence for exemplaryanti-STn CARs. SEQ ID NOs: 28-38 set forth the amino acid sequences ofvarious linkers.

SEQ ID NOs: 39-51 set forth the amino acid sequences of proteasecleavage sites and self-cleaving polypeptide cleavage sites.

SEQ ID NO: 52 sets forth the amino acid sequence for humantumor-associated glycoprotein (TAG-72).

SEQ ID NO: 53 sets forth the amino acid sequence of an exemplary rulefor determining a CDR-L3.

SEQ ID NO: 54 sets forth the amino acid sequence of an exemplary rulefor determining a CDR-H1.

SEQ ID NO: 55 sets forth the amino acid sequence of an exemplary rulefor determining a CDR-H2.

SEQ ID NO: 56 sets forth the amino acid sequence of an exemplary rulefor determining a CDR-H3.

SEQ ID NO: 57 sets forth the nucleotide sequence for the consensus Kozaksequence.

DETAILED DESCRIPTION A. Overview

The invention generally relates to improved compositions and methods forpreventing or treating cancers that express the STn antigen on aglycoprotein or preventing, treating, or ameliorating at least onesymptom associated with an STn expressing cancer. In particularembodiments, the invention relates to improved adoptive cell therapy ofcancers that express STn antigen on a glycoprotein using geneticallymodified immune effector cells. Genetic approaches offer a potentialmeans to enhance immune recognition and elimination of cancer cells. Onepromising strategy is to genetically engineer immune effector cells toexpress chimeric antigen receptors (CAR) that redirect cytotoxicitytoward cancer cells.

The improved compositions and methods of adoptive cell therapycontemplated herein, provide genetically modified immune effector cellsthat can readily be expanded, exhibit long-term persistence in vivo, anddemonstrate antigen dependent cytotoxicity to cells expressing sialyl Tnantigen (STn) on a glycoprotein.

STn (Neu5Acα2-6GalNAcα-O-Ser/Thr is also known as CD175s and is thesimplest sialylated mucin-type O-glycan. STn is a disaccharide formed ofone residue of N-acetyl-galactosamine (GalNAc) alpha-O-linked to aserine or a threonine residue, and substituted by a sialic acid (Neu5Acin human) on carbon 6. This sialylation prevents the formation ofvarious core structures otherwise found in mucin-type O-glycans.

STn is expressed in fetal tissues such as the esophagus, stomach,pancreas, colon (goblet cells), lung, mammary gland, and gonadal tissuesfrom fetuses of both sexes. However, not much is known about thebiological role of STn during embryonic development.

Various studies have been performed that report STn expression in normaltissues to be rare and/or low compared to cancer tissues.Immunohistochemistry demonstrated the STn overexpression in cancer cellscompared to matched healthy cells. Thus, STn was described as anonco-fetal antigen. STn neo-expression or over-expression was reportedin many epithelial cancers with highest frequencies in pancreas,bladder, lung, colorectal, and ovarian cancers. Early in carcinogenesisSTn was reported to be over-expressed in several epithelial benignlesions considered to be potential precursors of cancers, such asesophageal dysplastic squamous epithelia, gastric intestinal metaplasia,colonic moderate dysplasia, lung atypical adematous hyperplasia, breastductal hyperplasia, and apocrine metaplasia. STn expression was alsoreported in benign lesions in pancreas and ovaries, two tissues that aredevoid of STn expression in the healthy state.

There are also reports linking STn expression to inflammatory diseasesof the stomach (gastritis) or the colon (ulcerative colitis and Crohn'scolitis). In gastritis, STn was detected in 50-100% of the cases. Inulcerative colitis, de-O-acetylated STn was shown to be an independentmarker of the dysplasia-carcinoma sequence. De-O-acetylated STn was alsodetected in 44% of the cases of Crohn's colitis, another inflammatorydisease associated with colon cancer risks.

In various embodiments, CARs comprising anti-STn antibody sequences arehighly efficacious; undergo robust in vivo expansion; and recognizecancer cells that express STn on glycoproteins and show cytotoxicactivity against the STn expressing cancer cells. The STn antigen ishighly expressed in a wide variety of solid tumors including, but notlimited to esophageal cancer, lung cancer, ovarian cancer, cervicalcancer, pancreatic cancer, choleangiocarcinoma, gastric cancer, coloncancer, and breast cancer.

In one embodiment, a CAR comprising an anti-STn antibody or antigenbinding fragment, a transmembrane domain, and one or more intracellularsignaling domains is provided.

In one embodiment, an immune effector cell is genetically modified toexpress a CAR. T cells expressing a CAR are referred to herein as CAR Tcells or CAR modified T cells.

In various embodiments, genetically modified immune effector cells areadministered to a patient with cancer cells that express STn onglycoproteins including, but not limited to solid tumors andhematological malignancies.

The practice of the particular embodiments will employ, unless indicatedspecifically to the contrary, conventional methods of chemistry,biochemistry, organic chemistry, molecular biology, microbiology,recombinant DNA techniques, genetics, immunology, and cell biology thatare within the skill of the art, many of which are described below forthe purpose of illustration. Such techniques are explained fully in theliterature. See, e.g., Sambrook, et al., Molecular Cloning: A LaboratoryManual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual (2nd Edition, 1989); Maniatis et al., MolecularCloning: A Laboratory Manual (1982); Ausubel et al., Current Protocolsin Molecular Biology (John Wiley and Sons, updated July 2008); ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I &II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis ofComplex Genomes, (Academic Press, New York, 1992); Transcription andTranslation (B. Hames & S. Higgins, Eds., 1984); Perbal, A PracticalGuide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) CurrentProtocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies,E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology;as well as monographs in journals such as Advances in Immunology.

B. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of particular embodiments, preferred embodimentsof compositions, methods and materials are described herein. For thepurposes of the present disclosure, the following terms are definedbelow.

The articles “a,” “an,” and “the” are used herein to refer to one or tomore than one (i.e., to at least one, or to one or more) of thegrammatical object of the article. By way of example, “an element” meansone element or one or more elements.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives.

The term “and/or” should be understood to mean either one, or both ofthe alternatives.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range ofquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%,±2%, or ±1% about a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are present that materially affect the activity or action ofthe listed elements.

Reference throughout this specification to “one embodiment,” “anembodiment,” “a particular embodiment,” “a related embodiment,” “acertain embodiment,” “an additional embodiment,” or “a furtherembodiment” or combinations thereof means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments. It is also understoodthat the positive recitation of a feature in one embodiment, serves as abasis for excluding the feature in a particular embodiment.

STn (Neu5Acα2-6GalNAcα-O-Ser/Thr is also known as CD175s and is thesimplest sialylated mucin-type O-glycan. STn is a disaccharide formed ofone residue of N-acetyl-galactosamine (GalNAc) alpha-O-linked to aserine or a threonine residue, and substituted by a sialic acid (Neu5Acin human) on carbon 6. This sialylation prevents the formation ofvarious core structures otherwise found in mucin-type O-glycans.

In various embodiments, the STn antigen is expressed on a glycoproteinselected from the group consisting of mucins or mucin-likeglycoproteins.

In particular embodiments, the STn antigen is expressed on a mucinselected from the group consisting of: mucin 1 and mucin 16.

In certain embodiments, the STn antigen is expressed on a mucin-likeprotein.

In various embodiments, the mucin-like protein is TAG-72.

C. Chimeric Antigen Receptors

In various embodiments, genetically engineered receptors that redirectcytotoxicity of immune effector cells toward cancer cells an STn antigenare provided. These genetically engineered receptors referred to hereinas chimeric antigen receptors (CARs). CARs are molecules that combineantibody-based specificity for a desired antigen (e.g., STn expressed ona glycoprotein) with a T cell receptor-activating intracellular domainto generate a chimeric protein that exhibits a specific anti-STncellular immune activity. As used herein, the term, “chimeric,”describes being composed of parts of different proteins or DNAs fromdifferent origins.

In particular embodiments, CARs comprise an extracellular domain (alsoreferred to as a binding domain or antigen-specific binding domain) thatbinds to STn, a transmembrane domain, and an intracellular signalingdomain. Engagement of the anti-STn antigen binding domain of the CARwith STn bound glycoprotein, e.g., TAG-72, on the surface of a targetcell results in clustering of the CAR and delivers an activationstimulus to the CAR-containing cell. The main characteristic of CARs aretheir ability to redirect immune effector cell specificity, therebytriggering proliferation, cytokine production, phagocytosis orproduction of molecules that can mediate cell death of the targetantigen expressing cell in a major histocompatibility (MHC) independentmanner, exploiting the cell specific targeting abilities of monoclonalantibodies, soluble ligands or cell specific co-receptors.

In various embodiments, a CAR comprises an extracellular binding domainthat comprises an STn-specific binding domain; a transmembrane domain;one or more intracellular co-stimulatory signaling domains; and aprimary signaling domain.

In particular embodiments, a CAR comprises an extracellular bindingdomain that comprises an anti-STn antibody or antigen binding fragmentthereof; one or more hinge domains or spacer domains; a transmembranedomain including; one or more intracellular co-stimulatory signalingdomains; and a primary signaling domain.

1. Binding Domain

In particular embodiments, CARs comprise an extracellular binding domainthat comprises an anti-STn antibody or antigen binding fragment thereofthat specifically binds to a human STn bound glycoprotein expressed on atarget cell, e.g., a cancer cell. As used herein, the terms, “bindingdomain,” “extracellular domain,” “extracellular binding domain,”“antigen-specific binding domain,” and “extracellular antigen specificbinding domain,” are used interchangeably and provide a CAR with theability to specifically bind to the target antigen of interest, e.g.,STn. The binding domain may be derived either from a natural, synthetic,semi-synthetic, or recombinant source.

The terms “specific binding affinity” or “specifically binds” or“specifically bound” or “specific binding” or “specifically targets” asused herein, describe binding of an anti-STn antibody or antigen bindingfragment thereof (or a CAR comprising the same) to STn at greaterbinding affinity than background binding. A binding domain (or a CARcomprising a binding domain or a fusion protein containing a bindingdomain) “specifically binds” to an STn bound glycoprotein, such asTAG-72, if it binds to or associates with STn with an affinity or K_(a)(i.e., an equilibrium association constant of a particular bindinginteraction with units of 1/M) of, for example, greater than or equal toabout 10⁵ M⁻¹. In certain embodiments, a binding domain (or a fusionprotein thereof) binds to a target with a K_(a) greater than or equal toabout 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹,or 10¹³ M⁻¹. “High affinity” binding domains (or single chain fusionproteins thereof) refers to those binding domains with a K_(a) of atleast 10⁷M⁻¹, at least 10⁸M⁻¹, at least 10⁹M⁻¹, at least 10¹⁰ M⁻¹, atleast 10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 10¹³M⁻¹, or greater.

Alternatively, affinity may be defined as an equilibrium dissociationconstant (K_(d)) of a particular binding interaction with units of M(e.g., 10⁻⁵ M to 10⁻¹³ M, or less). Affinities of binding domainpolypeptides and CAR proteins according to the present disclosure can bereadily determined using conventional techniques, e.g., by competitiveELISA (enzyme-linked immunosorbent assay), or by binding association, ordisplacement assays using labeled ligands, or using a surface-plasmonresonance device such as the Biacore T100, which is available fromBiacore, Inc., Piscataway, N.J., or optical biosensor technology such asthe EPIC system or EnSpire that are available from Corning and PerkinElmer respectively (see also, e.g., Scatchard et al. (1949) Ann. N.Y.Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173; 5,468,614, or theequivalent).

In one embodiment, the affinity of specific binding is about 2 timesgreater than background binding, about 5 times greater than backgroundbinding, about 10 times greater than background binding, about 20 timesgreater than background binding, about 50 times greater than backgroundbinding, about 100 times greater than background binding, or about 1000times greater than background binding or more.

In particular embodiments, the extracellular binding domain of a CARcomprises an antibody or antigen binding fragment thereof. An “antibody”refers to a binding agent that is a polypeptide comprising at least alight chain or heavy chain immunoglobulin variable region whichspecifically recognizes and binds an epitope of an antigen, such as alipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleicacid containing an antigenic determinant, such as those recognized by animmune cell.

An “antigen (Ag)” refers to a compound, composition, or substance thatcan stimulate the production of antibodies or a T cell response in ananimal, including compositions (such as one that includes acancer-specific protein) that are injected or absorbed into an animal.Exemplary antigens include but are not limited to lipids, carbohydrates,polysaccharides, glycoproteins, peptides, or nucleic acids. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous antigens, such as the disclosedantigens. In particular embodiments, the target antigen is an STnantigen.

An “epitope” or “antigenic determinant” refers to the region of anantigen to which a binding agent binds.

Antibodies include antigen binding fragments thereof, such as Camel Ig,Ig NAR, Fab fragments, Fab′ fragments, F(ab)′₂ fragments, F(ab)′₃fragments, Fv, single chain Fv proteins (“scFv”), bis-scFv, (scFv)₂,minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fvproteins (“dsFv”), and single-domain antibody (sdAb, Nanobody) andportions of full length antibodies responsible for antigen binding. Theterm also includes genetically engineered forms such as chimericantibodies (for example, humanized murine antibodies), heteroconjugateantibodies (such as, bispecific antibodies) and antigen bindingfragments thereof. See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3_(rd) Ed.,W. H. Freeman & Co., New York, 1997.

As would be understood by the skilled person and as described elsewhereherein, a complete antibody comprises two heavy chains and two lightchains. Each heavy chain consists of a variable region and a first,second, and third constant region, while each light chain consists of avariable region and a constant region. Mammalian heavy chains areclassified as α, δ, ε, γ, and μ. Mammalian light chains are classifiedas X or x. Immunoglobulins comprising the α, δ, ε, γ, and μ heavy chainsare classified as immunoglobulin (Ig)A, IgD, IgE, IgG, and IgM. Thecomplete antibody forms a “Y” shape. The stem of the Y consists of thesecond and third constant regions (and for IgE and IgM, the fourthconstant region) of two heavy chains bound together and disulfide bonds(inter-chain) are formed in the hinge. Heavy chains γ, α and δ have aconstant region composed of three tandem (in a line) Ig domains, and ahinge region for added flexibility; heavy chains μ and ε have a constantregion composed of four immunoglobulin domains. The second and thirdconstant regions are referred to as “CH2 domain” and “CH3 domain”,respectively. Each arm of the Y includes the variable region and firstconstant region of a single heavy chain bound to the variable andconstant regions of a single light chain. The variable regions of thelight and heavy chains are responsible for antigen binding.

Light and heavy chain variable regions contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The CDRs can be definedor identified by conventional methods, such as by sequence according toKabat et al. (Wu, T T and Kabat, E. A., J Exp Med. 132(2):211-50,(1970); Borden, P. and Kabat E. A., PNAS, 84: 2440-2443 (1987); (see,Kabat et al., Sequences of Proteins of Immunological Interest, U.S.Department of Health and Human Services, 1991, which is herebyincorporated by reference), or by structure according to Chothia et al(Chothia, C. and Lesk, A. M., J Mol. Biol., 196(4): 901-917 (1987),Chothia, C. et al, Nature, 342: 877-883 (1989)).

Illustrative examples of rules for predicting light chain CDRs include:CDR-L1 starts at about residue 24, is preceded by a Cys, is about 10-17residues, and is followed by a Trp (typically Trp-Tyr-Gln, but also,Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu); CDR-L2 starts about 16 residuesafter the end of CDR-L1, is generally preceded by Ile-Tyr, but also,Val-Tyr, Ile-Lys, Ile-Phe, and is 7 residues; and CDR-L3 starts about 33residues after the end of CDR-L2, is preceded by a Cys, is 7-11residues, and is followed by Phe-Gly-XXX-Gly (XXX is any amino acid, SEQID NO: 53).

Illustrative examples of rules for predicting heavy chain CDRs include:CDR-H1 starts at about residue 26, is preceded by Cys-XXX-XXX-XXX (SEQID NO: 54), is 10-12 residues and is followed by a Trp (typicallyTrp-Val, but also, Trp-Ile, Trp-Ala); CDR-H2 starts about 15 residuesafter the end of CDR-H1, is generally preceded by Leu-Glu-Trp-Ile-Gly(SEQ ID NO: 55), or a number of variations, is 16-19 residues, and isfollowed by Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala; and CDR-H3starts about 33 residues after the end of CDR-H2, is preceded byCys-XXX-XXX (typically Cys-Ala-Arg), is 3 to 25 residues, and isfollowed by Trp-Gly-XXX-Gly (SEQ ID NO: 56).

In one embodiment, light chain CDRs and the heavy chain CDRs aredetermined according to the Kabat method

In one embodiment, light chain CDRs and the heavy chain CDR2 and CDR3are determined according to the Kabat method, and heavy chain CDR1 isdetermined according to the AbM method, which is a comprise between theKabat and Clothia methods, see e.g., Whitelegg N & Rees A R, ProteinEng. 2000 December; 13(12):819-24 and Methods Mol Biol. 2004; 248:51-91.Programs for predicting CDRs are publicly available, e.g., AbYsis(www.bioinf.org.uk/abysis/).

The sequences of the framework regions of different light or heavychains are relatively conserved within a species, such as humans. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs in three-dimensional space. The CDRs are primarily responsiblefor binding to an epitope of an antigen. The CDRs of each chain aretypically referred to as CDR1, CDR2, and CDR3, numbered sequentiallystarting from the N-terminus, and are also typically identified by thechain in which the particular CDR is located. Thus, the CDRs located inthe variable domain of the heavy chain of the antibody are referred toas CDRH1, CDRH2, and CDRH3, whereas the CDRs located in the variabledomain of the light chain of the antibody are referred to as CDRL1,CDRL2, and CDRL3. Antibodies with different specificities (i.e.,different combining sites for different antigens) have different CDRs.Although it is the CDRs that vary from antibody to antibody, only alimited number of amino acid positions within the CDRs are directlyinvolved in antigen binding. These positions within the CDRs are calledspecificity determining residues (SDRs). Illustrative examples of lightchain CDRs that are suitable for constructing anti-STn CARs contemplatedherein include, but are not limited to the CDR sequences set forth inSEQ ID NOs: 1-3, 9-11, and 17-19. Illustrative examples of heavy chainCDRs that are suitable for constructing anti-STn CARs contemplatedherein include, but are not limited to the CDR sequences set forth inSEQ ID NOs: 4-6, 12-14, and 20-22.

References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an antibody, Fv, scFv,dsFv, Fab, or other antibody fragment as disclosed herein. Illustrativeexamples of light chain variable regions that are suitable forconstructing anti-STn CARs contemplated herein include, but are notlimited to the light chain variable region sequences set forth in SEQ IDNOs: 7, 15, and 23.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an antibody, Fv, scFv,dsFv, Fab, or other antibody fragment as disclosed herein. Illustrativeexamples of heavy chain variable regions that are suitable forconstructing anti-STn CARs contemplated herein include, but are notlimited to the heavy chain variable region sequences set forth in SEQ IDNOs: 8, 16, and 24.

A “monoclonal antibody” is an antibody produced by a single clone of Blymphocytes or by a cell into which the light and heavy chain genes of asingle antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a mouse. In particular preferred embodiments, a CARcomprises antigen-specific binding domain that is a chimeric antibody orantigen binding fragment thereof.

In preferred embodiments, the antibody is a human antibody (such as ahuman monoclonal antibody) or fragment thereof that specifically bindsto a human STn expressing glycoprotein. Human antibodies can beconstructed by combining Fv clone variable domain sequence(s) selectedfrom human-derived phage display libraries with known human constantdomain sequences(s) as described above. Alternatively, human monoclonalantibodies may be made by the hybridoma method. Human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991). Inaddition, transgenic animals (e.g., mice) can be used to produce a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. See, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann etal., Year in Immunol., 7: 33 (1993). Gene shuffling can also be used toderive human antibodies from non-human, e.g., rodent antibodies, wherethe human antibody has similar affinities and specificities to thestarting non-human antibody. (see PCT WO 93/06213 published Apr. 1,1993). Unlike traditional humanization of non-human antibodies by CDRgrafting, this technique provides completely human antibodies, whichhave no FR or CDR residues of non-human origin.

In one embodiment, a CAR comprises a “humanized” antibody. A humanizedantibody is an immunoglobulin including a human framework region and oneor more CDRs from a non-human (for example a mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor,” and the human immunoglobulin providing the frameworkis termed an “acceptor.” In one embodiment, all the CDRs are from thedonor immunoglobulin in a humanized immunoglobulin. Constant regionsneed not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. Humanized or other monoclonal antibodies can have additionalconservative amino acid substitutions, which have substantially noeffect on antigen binding or other immunoglobulin functions. Humanizedantibodies can be constructed by means of genetic engineering (see forexample, U.S. Pat. No. 5,585,089).

In particular embodiments, an anti-STn antibody or antigen bindingfragment thereof, includes but is not limited to a Camel Ig (a camelidantibody (VHH)), Ig NAR, Fab fragments, Fab′ fragments, F(ab)′2fragments, F(ab)′3 fragments, Fv, single chain Fv antibody (“scFv”),bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfidestabilized Fv protein (“dsFv”), and single-domain antibody (sdAb,Nanobody).

“Camel Ig” or “camelid VHH” as used herein refers to the smallest knownantigen-binding unit of a heavy chain antibody (Koch-Nolte, et al, FASEBJ., 21: 3490-3498 (2007)). A “heavy chain antibody” or a “camelidantibody” refers to an antibody that contains two VH domains and nolight chains (Riechmann L. et al, J. Immunol. Methods 231:25-38 (1999);WO94/04678; WO94/25591; U.S. Pat. No. 6,005,079).

“IgNAR” of “immunoglobulin new antigen receptor” refers to class ofantibodies from the shark immune repertoire that consist of homodimersof one variable new antigen receptor (VNAR) domain and five constant newantigen receptor (CNAR) domains. IgNARs represent some of the smallestknown immunoglobulin-based protein scaffolds and are highly stable andpossess efficient binding characteristics. The inherent stability can beattributed to both (i) the underlying Ig scaffold, which presents aconsiderable number of charged and hydrophilic surface exposed residuescompared to the conventional antibody VH and VL domains found in murineantibodies; and (ii) stabilizing structural features in thecomplementary determining region (CDR) loops including inter-loopdisulfide bridges, and patterns of intra-loop hydrogen bonds.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three hypervariableregions (HVRs) of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six HVRs confer antigen-binding specificity to the antibody.However, even a single variable domain (or half of an Fv comprising onlythree HVRs specific for an antigen) has the ability to recognize andbind antigen, although at a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)2 antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., PNAS USA 90: 6444-6448 (1993). Triabodies andtetrabodies are also described in Hudson et al., Nat. Med. 9:129-134(2003).

“Single domain antibody” or “sdAb” or “nanobody” refers to an antibodyfragment that consists of the variable region of an antibody heavy chain(VH domain) or the variable region of an antibody light chain (VLdomain) (Holt, L., et al, Trends in Biotechnology, 21(11): 484-490).

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain and in either orientation (e.g., VL-VH or VH-VL).Generally, the scFv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains which enables the scFv to form the desiredstructure for antigen binding. For a review of scFv, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

In preferred embodiments, a CAR comprises antigen-specific bindingdomain that is an scFv and may be a murine, human or humanized scFv.Single chain antibodies may be cloned from the V region genes of ahybridoma specific for a desired target. The production of suchhybridomas has become routine. A technique which can be used for cloningthe variable region heavy chain (V_(H)) and variable region light chain(V_(L)) has been described, for example, in Orlandi et al., PNAS, 1989;86: 3833-3837.

In particular embodiments, the antigen-specific binding domain that isan scFv that binds a human glycoprotein that expresses STn.

Illustrative examples of variable light chains that are suitable forconstructing anti-STn CARs contemplated herein include, but are notlimited to the amino acid sequences set forth in SEQ ID NOs: 7, 15, and23.

Illustrative examples of variable heavy chains that are suitable forconstructing anti-STn CARs contemplated herein include, but are notlimited to the amino acid sequences set forth in SEQ ID NOs: 8, 16, and24.

An exemplary STn-specific binding domain is an immunoglobulin variableregion specific for STn that comprises at least one human frameworkregion. A “human framework region” refers to a wild type (i.e.,naturally occurring) framework region of a human immunoglobulin variableregion, an altered framework region of a human immunoglobulin variableregion with less than about 50% (e.g., preferably less than about 45%,40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) of the amino acids in theregion are deleted or substituted (e.g., with one or more amino acidresidues of a nonhuman immunoglobulin framework region at correspondingpositions), or an altered framework region of a nonhuman immunoglobulinvariable region with less than about 50% (e.g., less than 45%, 40%, 30%,25%, 20%, 15%, 10%, or 5%) of the amino acids in the region deleted orsubstituted (e.g., at positions of exposed residues and/or with one ormore amino acid residues of a human immunoglobulin framework region atcorresponding positions) so that, in one embodiment, immunogenicity isreduced.

In certain embodiments, a human framework region is a wild typeframework region of a human immunoglobulin variable region. In certainother embodiments, a human framework region is an altered frameworkregion of a human immunoglobulin variable region with amino aciddeletions or substitutions at one, two, three, four, five, six, seven,eight, nine, ten or more positions. In other embodiments, a humanframework region is an altered framework region of a non-humanimmunoglobulin variable region with amino acid deletions orsubstitutions at one, two, three, four, five, six, seven, eight, nine,ten or more positions.

In particular embodiments, an STn-specific binding domain comprises atleast one, two, three, four, five, six, seven or eight human frameworkregions (FR) selected from human light chain FR1, human heavy chain FR1,human light chain FR2, human heavy chain FR2, human light chain FR3,human heavy chain FR3, human light chain FR4, and human heavy chain FR4.

Human FRs that may be present in an STn-specific binding domains alsoinclude variants of the exemplary FRs provided herein in which one, two,three, four, five, six, seven, eight, nine, ten or more amino acids ofthe exemplary FRs have been substituted or deleted.

In certain embodiments, an STn-specific binding domain comprises (a) ahumanized light chain variable region that comprises a human light chainFR1, a human light chain FR2, a human light chain FR3, and a human lightchain FR4, and (b) a humanized heavy chain variable region thatcomprises a human heavy chain FR1, a human heavy chain FR2, a humanheavy chain FR3, and a human heavy chain FR4.

STn-specific binding domains provided herein also comprise one, two,three, four, five, or six CDRs. Such CDRs may be nonhuman CDRs oraltered nonhuman CDRs selected from CDRL1, CDRL2 and CDRL3 of the lightchain and CDRH1, CDRH2 and CDRH3 of the heavy chain. In certainembodiments, an STn-specific binding domain comprises (a) a light chainvariable region that comprises a light chain CDRL1, a light chain CDRL2,and a light chain CDRL3, and (b) a heavy chain variable region thatcomprises a heavy chain CDRH1, a heavy chain CDRH2, and a heavy chainCDRH3.

In one embodiment, an STn-specific binding domain comprises light chainCDR sequences set forth in SEQ ID NOs: 1-3, 9-11, or 17-19. In aparticular embodiment, an STn-specific binding domain comprises lightchain CDR sequences with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to thelight chain CDR sequences set forth in SEQ ID NOs: 1-3, 9-11, or 17-19.

In one embodiment, an STn-specific binding domain comprises heavy chainCDR sequences set forth in SEQ ID NOs: 4-6, 12-14, or 20-22. In aparticular embodiment, an STn-specific binding domain comprises heavychain CDR sequences with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to theheavy chain CDR sequences set forth in SEQ ID NOs: 4-6, 12-14, or 20-22.

References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an antibody, Fv, scFv,dsFv, Fab, or other antibody fragment as disclosed herein. Illustrativeexamples of light chain variable regions that are suitable forconstructing anti-STn CARs contemplated herein include, but are notlimited to the light chain variable region sequences set forth in SEQ IDNOs: 7, 15, or 23.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an antibody, Fv, scFv,dsFv, Fab, or other antibody fragment as disclosed herein. Illustrativeexamples of heavy chain variable regions that are suitable forconstructing anti-STn CARs contemplated herein include, but are notlimited to the heavy chain variable region sequences set forth in SEQ IDNOs: 8, 16, or 24.

2. Linkers

In certain embodiments, the CARs comprise linker residues between thevarious domains, e.g., between V_(H) and V_(L) domains, added forappropriate spacing and conformation of the molecule. In particularembodiments the linker is a variable region linking sequence. A“variable region linking sequence,” is an amino acid sequence thatconnects the V_(H) and V_(L) domains and provides a spacer functioncompatible with interaction of the two sub-binding domains so that theresulting polypeptide retains a specific binding affinity to the sametarget molecule as an antibody that comprises the same light and heavychain variable regions. In particular embodiments, a linker separatesone or more heavy or light chain variable domains, hinge domains,transmembrane domains, co-stimulatory domains, and/or primary signalingdomains. CARs comprise one, two, three, four, or five or more linkers.In particular embodiments, the length of a linker is about 1 to about 25amino acids, about 5 to about 20 amino acids, or about 10 to about 20amino acids, or any intervening length of amino acids. In someembodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acidslong.

Illustrative examples of linkers include glycine polymers (G)_(n);glycine-serine polymers (G₁₋₅S₁₋₅)_(n), where n is an integer of atleast one, two, three, four, or five; glycine-alanine polymers;alanine-serine polymers; and other flexible linkers known in the art.Glycine and glycine-serine polymers are relatively unstructured, andtherefore may be able to serve as a neutral tether between domains offusion proteins such as the CARs described herein. Glycine accessessignificantly more phi-psi space than even alanine, and is much lessrestricted than residues with longer side chains (see Scheraga, Rev.Computational Chem. 11173-142 (1992)). The ordinarily skilled artisanwill recognize that design of a CAR in particular embodiments caninclude linkers that are all or partially flexible, such that the linkercan include a flexible linker as well as one or more portions thatconfer less flexible structure to provide for a desired CAR structure.

Other exemplary linkers include, but are not limited to the followingamino acid sequences: GGG; DGGGS (SEQ ID NO: 28); TGEKP (SEQ ID NO: 29)(see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 30)(Pomerantz et al. 1995, supra); (GGGGS)_(n) wherein=1, 2, 3, 4 or 5 (SEQID NO: 31) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQID NO: 32) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A.87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 33) (Bird et al., 1988,Science 242:423-426), GGRRGGGS (SEQ ID NO: 34); LRQRDGERP (SEQ ID NO:35); LRQKDGGGSERP (SEQ ID NO: 36); LRQKD(GGGS)₂ ERP (SEQ ID NO: 37).Alternatively, flexible linkers can be rationally designed using acomputer program capable of modeling both DNA-binding sites and thepeptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS91:11099-11103 (1994) or by phage display methods. In one embodiment,the linker comprises the following amino acid sequence:GSTSGSGKPGSGEGSTKG (SEQ ID NO: 38) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

3. Spacer Domain

In particular embodiments, the binding domain of the CAR is followed byone or more “spacer domains,” which refers to the region that moves theantigen binding domain away from the effector cell surface to enableproper cell/cell contact, antigen binding and activation (Patel et al.,Gene Therapy, 1999; 6: 412-419). The spacer domain may be derived eitherfrom a natural, synthetic, semi-synthetic, or recombinant source. Incertain embodiments, a spacer domain is a portion of an immunoglobulin,including, but not limited to, one or more heavy chain constant regions,e.g., CH2 and CH3. The spacer domain can include the amino acid sequenceof a naturally occurring immunoglobulin hinge region or an alteredimmunoglobulin hinge region.

In one embodiment, the spacer domain comprises the CH2 and CH3 domainsof IgG1, IgG4, or IgD.

4. Hinge Domain

The binding domain of the CAR is generally followed by one or more“hinge domains,” which plays a role in positioning the antigen bindingdomain away from the effector cell surface to enable proper cell/cellcontact, antigen binding and activation. A CAR generally comprises oneor more hinge domains between the binding domain and the transmembranedomain (TM). The hinge domain may be derived either from a natural,synthetic, semi-synthetic, or recombinant source. The hinge domain caninclude the amino acid sequence of a naturally occurring immunoglobulinhinge region or an altered immunoglobulin hinge region.

An “altered hinge region” refers to (a) a naturally occurring hingeregion with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%,10%, or 5% amino acid substitutions or deletions), (b) a portion of anaturally occurring hinge region that is at least 10 amino acids (e.g.,at least 12, 13, 14 or 15 amino acids) in length with up to 30% aminoacid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acidsubstitutions or deletions), or (c) a portion of a naturally occurringhinge region that comprises the core hinge region (which may be 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 amino acids in length). In certain embodiments,one or more cysteine residues in a naturally occurring immunoglobulinhinge region may be substituted by one or more other amino acid residues(e.g., one or more serine residues). An altered immunoglobulin hingeregion may alternatively or additionally have a proline residue of awild type immunoglobulin hinge region substituted by another amino acidresidue (e.g., a serine residue).

Illustrative hinge domains suitable for use in the CARs described hereininclude the hinge region derived from the extracellular regions of type1 membrane proteins such as CD8α, and CD4, which may be wild-type hingeregions from these molecules or may be altered. In another embodiment,the hinge domain comprises a CD8a hinge region.

In one embodiment, the hinge is a PD-1 hinge or CD152 hinge.

5. Transmembrane (TM) Domain

The “transmembrane domain” is the portion of the CAR that fuses theextracellular binding portion and intracellular signaling domain andanchors the CAR to the plasma membrane of the immune effector cell. TheTM domain may be derived either from a natural, synthetic,semi-synthetic, or recombinant source. The TM domain may be derived from(i.e., comprise at least the transmembrane region(s) of the alpha orbeta chain of the T-cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α,CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In a particular embodiment, the TMdomain is synthetic and predominantly comprises hydrophobic residuessuch as leucine and valine.

In one embodiment, the CARs comprise a TM domain derived from, PD1,CD152, or CD8α. In another embodiment, a CAR comprises a TM domainderived from, PD1, CD152, or CD8a and a short oligo- or polypeptidelinker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsin length that links the TM domain and the intracellular signalingdomain of the CAR. A glycine-serine based linker provides a particularlysuitable linker.

6. Intracellular Signaling Domain

In particular embodiments, CARs comprise an intracellular signalingdomain. An “intracellular signaling domain,” refers to the part of a CARthat participates in transducing the message of effective anti-STn CARbinding to a human TAG-72 polypeptide into the interior of the immuneeffector cell to elicit effector cell function, e.g., activation,cytokine production, proliferation and cytotoxic activity, including therelease of cytotoxic factors to the CAR-bound target cell, or othercellular responses elicited with antigen binding to the extracellularCAR domain.

The term “effector function” refers to a specialized function of animmune effector cell. Effector function of the T cell, for example, maybe cytolytic activity or help or activity including the secretion of acytokine. Thus, the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal andthat directs the cell to perform a specialized function. While usuallythe entire intracellular signaling domain can be employed, in many casesit is not necessary to use the entire domain. To the extent that atruncated portion of an intracellular signaling domain is used, suchtruncated portion may be used in place of the entire domain as long asit transduces the effector function signal. The term intracellularsignaling domain is meant to include any truncated portion of theintracellular signaling domain sufficient to transducing effectorfunction signal.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of intracellular signalingdomains: primary signaling domains that initiate antigen-dependentprimary activation through the TCR (e.g., a TCR/CD3 complex) andco-stimulatory signaling domains that act in an antigen-independentmanner to provide a secondary or co-stimulatory signal. In preferredembodiments, a CAR comprises an intracellular signaling domain thatcomprises one or more “co-stimulatory signaling domain” and a “primarysignaling domain.”

Primary signaling domains regulate primary activation of the TCR complexeither in a stimulatory way, or in an inhibitory way. Primary signalingdomains that act in a stimulatory manner may contain signaling motifswhich are known as immunoreceptor tyrosine-based activation motifs orITAMs.

Illustrative examples of ITAM containing primary signaling domains thatare suitable for use in particular embodiments include those derivedfrom FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.In particular preferred embodiments, a CAR comprises a CD3ζ primarysignaling domain and one or more co-stimulatory signaling domains. Theintracellular primary signaling and co-stimulatory signaling domains maybe linked in any order in tandem to the carboxyl terminus of thetransmembrane domain.

In particular embodiments, CARs comprise one or more co-stimulatorysignaling domains to enhance the efficacy and expansion of T cellsexpressing CAR receptors. As used herein, the term, “co-stimulatorysignaling domain,” or “co-stimulatory domain”, refers to anintracellular signaling domain of a co-stimulatory molecule.Co-stimulatory molecules are cell surface molecules other than antigenreceptors or Fc receptors that provide a second signal required forefficient activation and function of T lymphocytes upon binding toantigen. Illustrative examples of such co-stimulatory molecules includeTLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11,CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137(4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In oneembodiment, a CAR comprises one or more co-stimulatory signaling domainsselected from the group consisting of CD28, CD137, and CD134, and a CD3ζprimary signaling domain.

In another embodiment, a CAR comprises CD28 and CD137 co-stimulatorysignaling domains and a CD3ζ primary signaling domain.

In yet another embodiment, a CAR comprises CD28 and CD134 co-stimulatorysignaling domains and a CD3ζ primary signaling domain.

In one embodiment, a CAR comprises CD137 and CD134 co-stimulatorysignaling domains and a CD3ζ primary signaling domain.

In one embodiment, a CAR comprises a CD137 co-stimulatory signalingdomain and a CD3ζ primary signaling domain.

In one embodiment, a CAR comprises a CD134 co-stimulatory signalingdomain and a CD3ζ primary signaling domain.

In one embodiment, a CAR comprises a CD28 co-stimulatory signalingdomain and a CD3ζ primary signaling domain.

In particular embodiments, CARs comprise an anti-STn antibody or antigenbinding fragment thereof that specifically binds to a glycoproteinexpressing STn on a cancer cell.

In one embodiment, a CAR comprises an anti-STn scFv that binds STnexpressed on a glycoprotein; a transmembrane domain derived from apolypeptide selected from the group consisting of: alpha or beta chainof the T-cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137,CD152, CD154, and PD1; and one or more intracellular co-stimulatorysignaling domains from a co-stimulatory molecule selected from the groupconsisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83,CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76,TRIM, and ZAP70; and a primary signaling domain from FcRγ, FcRβ, CD3γ,CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

In one embodiment, a CAR comprises an anti-STn scFv that binds STnexpressed on a glycoprotein; a hinge domain selected from the groupconsisting of: IgG1 hinge/CH2/CH3, IgG4 hinge/CH2/CH3, a PD1 hinge, aCD152 hinge, and a CD8a hinge; a transmembrane domain derived from apolypeptide selected from the group consisting of: alpha or beta chainof the T-cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137,CD152, CD154, and PD1; and one or more intracellular co-stimulatorysignaling domains from a co-stimulatory molecule selected from the groupconsisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83,CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76,TRIM, and ZAP70; and a primary signaling domain from FcRγ, FcRβ, CD3γ,CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

In one embodiment, a CAR comprises an anti-STn scFv binds STn expressedon a glycoprotein; a hinge domain selected from the group consisting of:IgG1 hinge/CH2/CH3, IgG4 hinge/CH2/CH3, a PD1 hinge, a CD152 hinge, anda CD8a hinge; a transmembrane domain derived from a polypeptide selectedfrom the group consisting of: alpha or beta chain of the T-cellreceptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27,CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154,and PD1; a short oligo- or polypeptide linker, preferably between 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length that links the TMdomain to the intracellular signaling domain of the CAR; and one or moreintracellular co-stimulatory signaling domains from a co-stimulatorymolecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30,CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS),DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70; and a primary signalingdomain from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, andCD66d.

In a particular embodiment, a CAR comprises an anti-STn scFv that bindsSTn expressed on a glycoprotein; a hinge domain comprising an IgG1hinge/CH2/CH3 polypeptide and a CD8α polypeptide; a CD8α transmembranedomain comprising a polypeptide linker of about 3 to about 10 aminoacids; a CD137 intracellular co-stimulatory signaling domain; and a CD3ζprimary signaling domain.

In a particular embodiment, a CAR comprises an anti-STn scFv that bindsSTn expressed on a glycoprotein; a hinge domain comprising a CD8αpolypeptide; a CD8α transmembrane domain comprising a polypeptide linkerof about 3 to about 10 amino acids; a CD134 intracellular co-stimulatorysignaling domain; and a CD3ζ primary signaling domain.

In a particular embodiment, a CAR comprises an anti-STn scFv that bindsSTn expressed on a glycoprotein; a hinge domain comprising a CD8αpolypeptide; a CD8α transmembrane domain comprising a polypeptide linkerof about 3 to about 10 amino acids; a CD28 intracellular co-stimulatorysignaling domain; and a CD3ζ primary signaling domain.

In a particular embodiment, a CAR comprises an anti-STn scFv that bindsSTn expressed on a glycoprotein; a hinge domain comprising a PD1 hinge,polypeptide; a PD1 or CD152 transmembrane domain comprising apolypeptide linker of about 3 to about 10 amino acids; a CD137intracellular co-stimulatory signaling domain; and a CD3ζ primarysignaling domain.

In a particular embodiment, a CAR comprises an anti-STn scFv that bindsSTn expressed on a glycoprotein; a hinge domain comprising a PD1 hinge,polypeptide; a PD1 or CD152 transmembrane domain comprising apolypeptide linker of about 3 to about 10 amino acids; a CD134intracellular co-stimulatory signaling domain; and a CD3ζ primarysignaling domain.

In a particular embodiment, a CAR comprises an anti-STn scFv that bindsSTn expressed on a glycoprotein; a hinge domain comprising a PD1 hinge,polypeptide; a PD1 or CD152 transmembrane domain comprising apolypeptide linker of about 3 to about 10 amino acids; a CD28intracellular co-stimulatory signaling domain; and a CD3ζ primarysignaling domain.

In a particular embodiment, a CAR comprises an anti-STn scFv that bindsSTn expressed on a glycoprotein; a hinge domain comprising a CD152hinge, polypeptide; a PD1 or CD152 transmembrane domain comprising apolypeptide linker of about 3 to about 10 amino acids; a CD137intracellular co-stimulatory signaling domain; and a CD3 primarysignaling domain.

In a particular embodiment, a CAR comprises an anti-STn scFv that bindsSTn expressed on a glycoprotein; a hinge domain comprising a CD152hinge, polypeptide; a PD1 or CD152 transmembrane domain comprising apolypeptide linker of about 3 to about 10 amino acids; a CD134intracellular co-stimulatory signaling domain; and a CD3 primarysignaling domain.

In a particular embodiment, a CAR comprises an anti-STn scFv that bindsSTn expressed on a glycoprotein; a hinge domain comprising a CD152hinge, polypeptide; a PD1 or CD152 transmembrane domain comprising apolypeptide linker of about 3 to about 10 amino acids; a CD28intracellular co-stimulatory signaling domain; and a CD3ζ primarysignaling domain.

Moreover, the design of the CARs contemplated herein enable improvedexpansion, long-term persistence, and cytotoxic properties in T cellsexpressing the CARs compared to non-modified T cells or T cells modifiedto express other CARs.

D. Polypeptides

Various polypeptides are contemplated herein, including, but not limitedto, CAR polypeptides and fragments thereof, cells and compositionscomprising the same, and vectors that express polypeptides. In preferredembodiments, a polypeptide comprising one or more CARs is provided. Inparticular embodiments, the CAR is an anti-STn CAR comprising an aminoacid sequence as set forth in any one of SEQ ID NOs: 25-27.

“Polypeptide,” “polypeptide fragment,” “peptide” and “protein” are usedinterchangeably, unless specified to the contrary, and according toconventional meaning, i.e., as a sequence of amino acids. Polypeptidesmay be synthesized or recombinantly produced. Polypeptides are notlimited to a specific length, e.g., they may comprise a full lengthprotein sequence or a fragment of a full length protein, and may includepost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. In various embodiments, the CAR polypeptidescomprise a signal (or leader) sequence at the N-terminal end of theprotein, which co-translationally or post-translationally directstransfer of the protein. Illustrative examples of suitable signalsequences useful in CARs contemplated herein include, but are notlimited to the IgG1 heavy chain signal polypeptide, a CD8α signalpolypeptide, or a human GM-C SF receptor alpha signal polypeptide.Polypeptides can be prepared using any of a variety of well-knownrecombinant and/or synthetic techniques. Polypeptides contemplatedherein specifically encompass the CARs of the present disclosure, orsequences that have deletions from, additions to, and/or substitutionsof one or more amino acid of a CAR as contemplated herein.

An “isolated peptide” or an “isolated polypeptide” and the like, as usedherein, refer to in vitro isolation and/or purification of a peptide orpolypeptide molecule from a cellular environment, and from associationwith other components of the cell, i.e., it is not significantlyassociated with in vivo substances. Similarly, an “isolated cell” refersto a cell that has been obtained from an in vivo tissue or organ and issubstantially free of extracellular matrix.

Polypeptides include “polypeptide variants.” Polypeptide variants maydiffer from a naturally occurring polypeptide in one or moresubstitutions, deletions, additions and/or insertions. Such variants maybe naturally occurring or may be synthetically generated, for example,by modifying one or more of the above polypeptide sequences. Forexample, in particular embodiments, it may be desirable to improve thebinding affinity and/or other biological properties of the CARs byintroducing one or more substitutions, deletions, additions and/orinsertions into a binding domain, hinge, TM domain, co-stimulatorysignaling domain or primary signaling domain of a CAR polypeptide. Inparticular embodiments, polypeptides include polypeptides having atleast about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity toany of the reference sequences contemplated herein, typically where thevariant maintains at least one biological activity of the referencesequence.

Polypeptides include “polypeptide fragments.” Polypeptide fragmentsrefer to a polypeptide, which can be monomeric or multimeric that has anamino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion or substitution of a naturally-occurring orrecombinantly-produced polypeptide. In certain embodiments, apolypeptide fragment can comprise an amino acid chain at least 5 toabout 500 amino acids long. It will be appreciated that in certainembodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350,400, or 450 amino acids long. Particularly useful polypeptide fragmentsinclude functional domains, including antigen-binding domains orfragments of antibodies. In the case of an anti-STn antibody, usefulfragments include, but are not limited to: a CDR region, a CDR3 regionof the heavy or light chain; a variable region of a heavy or lightchain; a portion of an antibody chain or variable region including twoCDRs; and the like.

The polypeptide may also be fused in-frame or conjugated to a linker orother sequence for ease of synthesis, purification or identification ofthe polypeptide (e.g., poly-His), or to enhance binding of thepolypeptide to a solid support.

As noted above, polypeptides may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a reference polypeptide can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkel(1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987,Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J.D. et al., (Molecular Biology of the Gene, Fourth Edition,Benjamin/Cummings, Menlo Park, Calif., 1987) and the references citedtherein. Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al., (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found., Washington, D.C.).

In certain embodiments, a polypeptide variant comprises one or moreconservative substitutions. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged. Modifications may be madein the structure of the polynucleotides and polypeptides contemplated inparticular embodiments and still obtain a functional molecule thatencodes a variant or derivative polypeptide with desirablecharacteristics. When it is desired to alter the amino acid sequence ofa polypeptide to create an equivalent, or even an improved, variantpolypeptide, one skilled in the art, for example, can change one or moreof the codons of the encoding DNA sequence, e.g., according to Table 1.

TABLE 1 Amino Acid Codons One Three letter letter Amino Acids code codeCodons Alanine A Ala GCA GCC GCG GCU Cysteine C Cys UGC UGU Asparticacid D Asp GAC GAU Glutamic acid E Glu GAA GAG Phenylalanine F Phe UUCUUU Glycine G Gly GGA GGC GGG GGU Histidine H His CAC CAU Isoleucine IIso AUA AUC AUU Lysine K Lys AAA AAG Leucine L Leu UUA UUG CUA CUC CUGCUU Methionine M Met AUG Asparagine N Asn AAC AAU Proline P Pro CCA CCCCCG CCU Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGUSerine S Ser AGC AGU UCA UCC UCG UCU Threonine T Thr ACA ACC ACG ACUValine V Val GUA GUC GUG GUU Tryptophan W Trp UGG Tyrosine Y Tyr UAC UAU

Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological activity can be foundusing computer programs well known in the art, such as DNASTAR, DNAStrider, Geneious, Mac Vector, or Vector NTI software. Preferably, aminoacid changes in the protein variants disclosed herein are conservativeamino acid changes, i.e., substitutions of similarly charged oruncharged amino acids. A conservative amino acid change involvessubstitution of one of a family of amino acids which are related intheir side chains. Naturally occurring amino acids are generally dividedinto four families: acidic (aspartate, glutamate), basic (lysine,arginine, histidine), non-polar (alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), and uncharged polar(glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine)amino acids. Phenylalanine, tryptophan, and tyrosine are sometimesclassified jointly as aromatic amino acids. In a peptide or protein,suitable conservative substitutions of amino acids are known to those ofskill in this art and generally can be made without altering abiological activity of a resulting molecule. Those of skill in this artrecognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson et al. Molecular Biology of theGene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p. 224).

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics (Kyte andDoolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions may be based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like.

Polypeptide variants further include glycosylated forms, aggregativeconjugates with other molecules, and covalent conjugates with unrelatedchemical moieties (e.g., pegylated molecules). Covalent variants can beprepared by linking functionalities to groups which are found in theamino acid chain or at the N- or C-terminal residue, as is known in theart. Variants also include allelic variants, species variants, andmuteins. Truncations or deletions of regions which do not affectfunctional activity of the proteins are also variants.

In one embodiment, where expression of two or more polypeptides isdesired, the polynucleotide sequences encoding them can be separated byan IRES sequence as discussed elsewhere herein. In another embodiment,two or more polypeptides can be expressed as a fusion protein thatcomprises one or more self-cleaving polypeptide sequences.

Polypeptides contemplated in particular embodiments include fusionpolypeptides. In preferred embodiments, fusion polypeptides andpolynucleotides encoding fusion polypeptides are provided, e.g., CARs.Fusion polypeptides and fusion proteins refer to a polypeptide having atleast two, three, four, five, six, seven, eight, nine, or ten or morepolypeptide segments. Fusion polypeptides are typically linkedC-terminus to N-terminus, although they can also be linked C-terminus toC-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. Thepolypeptides of the fusion protein can be in any order or a specifiedorder. Fusion polypeptides or fusion proteins can also includeconservatively modified variants, polymorphic variants, alleles,mutants, subsequences, and interspecies homologs, so long as the desiredtranscriptional activity of the fusion polypeptide is preserved. Fusionpolypeptides may be produced by chemical synthetic methods or bychemical linkage between the two moieties or may generally be preparedusing other standard techniques. Ligated DNA sequences comprising thefusion polypeptide are operably linked to suitable transcriptional ortranslational control elements as discussed elsewhere herein.

In one embodiment, a fusion partner comprises a sequence that assists inexpressing the protein (an expression enhancer) at higher yields thanthe native recombinant protein. Other fusion partners may be selected soas to increase the solubility of the protein or to enable the protein tobe targeted to desired intracellular compartments or to facilitatetransport of the fusion protein through the cell membrane.

Fusion polypeptides may further comprise a polypeptide cleavage signalbetween each of the polypeptide domains described herein. In addition, apolypeptide cleavage site can be put into any linker peptide sequence.Exemplary polypeptide cleavage signals include polypeptide cleavagerecognition sites such as protease cleavage sites, nuclease cleavagesites (e.g., rare restriction enzyme recognition sites, self-cleavingribozyme recognition sites), and self-cleaving viral oligopeptides (seedeFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

Suitable protease cleavages sites and self-cleaving peptides are knownto the skilled person (see, e.g., in Ryan et al., 1997. J. Gener. Virol.78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).Exemplary protease cleavage sites include, but are not limited to thecleavage sites of potyvirus NIa proteases (e.g., tobacco etch virusprotease), potyvirus HC proteases, potyvirus P1 (P35) proteases,byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus Lproteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3Cproteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (ricetungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleckvirus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.Due to its high cleavage stringency, TEV (tobacco etch virus) proteasecleavage sites are preferred in one embodiment, e.g., EXXYXQ(G/S) (SEQID NO: 39), for example, ENLYFQG (SEQ ID NO: 40) and ENLYFQS (SEQ ID NO:41), wherein X represents any amino acid (cleavage by TEV occurs betweenQ and G or Q and S).

In a particular embodiment, self-cleaving peptides include thosepolypeptide sequences obtained from potyvirus and cardiovirus 2Apeptides, FMDV (foot-and-mouth disease virus), equine rhinitis A virus,Thosea asigna virus and porcine teschovirus.

In certain embodiments, the self-cleaving polypeptide site comprises a2A or 2A-like site, sequence or domain (Donnelly et al., 2001. J. Gen.Virol. 82:1027-1041). Exemplary 2A sites are shown in Table 2.

TABLE 2 SEQ ID NO: 42 LLNFDLLKLAGDVESNPGP SEQ ID NO: 43TLNFDLLKLAGDVESNPGP SEQ ID NO: 44 LLKLAGDVESNPGP SEQ ID NO: 45NFDLLKLAGDVESNPGP SEQ ID NO: 46 QLLNFDLLKLAGDVESNPGP SEQ ID NO: 47APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 48VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIV APVKQT SEQ ID NO: 49LNFDLLKLAGDVESNPGP SEQ ID NO: 50 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 51 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

In preferred embodiments, a polypeptide comprises a CAR polypeptide.

E. Polynucleotides

In preferred embodiments, a polynucleotide encoding one or more CARpolypeptides is provided. As used herein, the terms “polynucleotide” or“nucleic acid” refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA),plus strand RNA (RNA(+)), minus strand RNA (RNA(−)), genomic DNA (gDNA),complementary DNA (cDNA) or recombinant DNA. Polynucleotides includesingle and double stranded polynucleotides. In particular embodiments,polynucleotides include polynucleotides or variants having at leastabout 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequenceidentity to any of the reference sequences contemplated herein. Invarious illustrative embodiments, polynucleotides include expressionvectors, viral vectors, and transfer plasmids, and compositions andcells comprising the same. In various illustrative embodiments,polynucleotides encode a polypeptide contemplated herein, including, butnot limited to the polypeptide sequences set forth in SEQ ID NOs: 1-51.

In particular embodiments, polynucleotides are provided that encode atleast about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000,1250, 1500, 1750, or 2000 or more contiguous amino acid residues of apolypeptide, as well as all intermediate lengths. It will be readilyunderstood that “intermediate lengths,” in this context, means anylength between the quoted values, such as 6, 7, 8, 9, etc., 101, 102,103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

As used herein, the terms “polynucleotide variant” and “variant” and thelike refer to polynucleotides displaying substantial sequence identitywith a reference polynucleotide sequence or polynucleotides thathybridize with a reference sequence under stringent conditions that aredefined hereinafter. These terms include polynucleotides in which one ormore nucleotides have been added or deleted, or replaced with differentnucleotides compared to a reference polynucleotide. In this regard, itis well understood in the art that certain alterations inclusive ofmutations, additions, deletions and substitutions can be made to areference polynucleotide whereby the altered polynucleotide retains thebiological function or activity of the reference polynucleotide.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Included are nucleotides and polypeptides having at leastabout 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequenceidentity to any of the reference sequences described herein, typicallywhere the polypeptide variant maintains at least one biological activityof the reference polypeptide.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity,” and “substantial identity”. A “reference sequence” is atleast 12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons Inc, 1994-1998, Chapter 15.

As used herein, “isolated polynucleotide” refers to a polynucleotidethat has been purified from the sequences which flank it in anaturally-occurring state, e.g., a DNA fragment that has been removedfrom the sequences that are normally adjacent to the fragment. An“isolated polynucleotide” also refers to a complementary DNA (cDNA), arecombinant DNA, or other polynucleotide that does not exist in natureand that has been made by the hand of man.

Terms that describe the orientation of polynucleotides include: 5′(normally the end of the polynucleotide having a free phosphate group)and 3′ (normally the end of the polynucleotide having a free hydroxyl(OH) group). Polynucleotide sequences can be annotated in the 5′ to 3′orientation or the 3′ to 5′ orientation. For DNA and mRNA, the 5′ to 3′strand is designated the “sense,” “plus,” or “coding” strand because itssequence is identical to the sequence of the premessenger (premRNA)[except for uracil (U) in RNA, instead of thymine (T) in DNA]. For DNAand mRNA, the complementary 3′ to 5′ strand which is the strandtranscribed by the RNA polymerase is designated as “template,”“antisense,” “minus,” or “non-coding” strand. As used herein, the term“reverse orientation” refers to a 5′ to 3′ sequence written in the 3′ to5′ orientation or a 3′ to 5′ sequence written in the 5′ to 3′orientation.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the complementary strand of the DNA sequence 5′ A G T C A T G3′ is 3′ T C A G T A C 5′. The latter sequence is often written as thereverse complement with the 5′ end on the left and the 3′ end on theright, 5′ C A T G A C T 3′. A sequence that is equal to its reversecomplement is said to be a palindromic sequence. Complementarity can be“partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there can be “complete” or“total” complementarity between the nucleic acids.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide, or fragment of variantthereof, as described herein. Some of these polynucleotides bear minimalhomology to the nucleotide sequence of any native gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated in particular embodiments, for examplepolynucleotides that are optimized for human and/or primate codonselection. In particular embodiments, the polynucleotides are codonoptimized for expression and/or stability. Further, alleles of the genescomprising the polynucleotide sequences provided herein may also beused. Alleles are endogenous genes that are altered as a result of oneor more mutations, such as deletions, additions and/or substitutions ofnucleotides.

The term “nucleic acid cassette” as used herein refers to geneticsequences within a vector which can express a RNA, and subsequently aprotein. The nucleic acid cassette contains the gene of interest, e.g.,a CAR. The nucleic acid cassette is positionally and sequentiallyoriented within the vector such that the nucleic acid in the cassettecan be transcribed into RNA, and when necessary, translated into aprotein or a polypeptide, undergo appropriate post-translationalmodifications required for activity in the transformed cell, and betranslocated to the appropriate compartment for biological activity bytargeting to appropriate intracellular compartments or secretion intoextracellular compartments. Preferably, the cassette has its 3′ and 5′ends adapted for ready insertion into a vector, e.g., it has restrictionendonuclease sites at each end. In a preferred embodiment, the nucleicacid cassette contains the sequence of a chimeric antigen receptor usedto increase the cytotoxicity of cancer cells that express an STn boundglycoprotein, e.g., TAG-72. The cassette can be removed and insertedinto a plasmid or viral vector as a single unit.

In particular embodiments, polynucleotides include at least onepolynucleotide-of-interest. As used herein, the term“polynucleotide-of-interest” refers to a polynucleotide encoding apolypeptide (i.e., a polypeptide-of-interest), inserted into anexpression vector that is desired to be expressed. A vector may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides-of-interest. In certainembodiments, the polynucleotide-of-interest encodes a polypeptide thatprovides a therapeutic effect in the treatment or prevention of adisease or disorder. Polynucleotides-of-interest, and polypeptidesencoded therefrom, include both polynucleotides that encode wild-typepolypeptides, as well as functional variants and fragments thereof. Inparticular embodiments, a functional variant has at least 80%, at least90%, at least 95%, or at least 99% identity to a corresponding wild-typereference polynucleotide or polypeptide sequence. In certainembodiments, a functional variant or fragment has at least 50%, at least60%, at least 70%, at least 80%, or at least 90% of a biologicalactivity of a corresponding wild-type polypeptide.

In one embodiment, the polynucleotide-of-interest is a template totranscribe miRNA, siRNA, or shRNA, ribozyme, or other inhibitory RNA. Invarious other embodiments, a polynucleotide comprises apolynucleotide-of-interest encoding a CAR and one or more additionalpolynucleotides-of-interest including, but not limited to, an inhibitorynucleic acid sequence including, without limitation: an siRNA, an miRNA,an shRNA, and a ribozyme.

As used herein, the terms “siRNA” or “short interfering RNA” refer to ashort polynucleotide sequence that mediates a process ofsequence-specific post-transcriptional gene silencing, translationalinhibition, transcriptional inhibition, or epigenetic RNAi in animals(Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391,806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999,Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13, 139-141; andStrauss, 1999, Science, 286, 886). In certain embodiments, an siRNAcomprises a first strand and a second strand that have the same numberof nucleosides; however, the first and second strands are offset suchthat the two terminal nucleosides on the first and second strands arenot paired with a residue on the complimentary strand. In certaininstances, the two nucleosides that are not paired are thymidineresides. The siRNA should include a region of sufficient homology to thetarget gene, and be of sufficient length in terms of nucleotides, suchthat the siRNA, or a fragment thereof, can mediate down regulation ofthe target gene. Thus, an siRNA includes a region which is at leastpartially complementary to the target RNA. It is not necessary thatthere be perfect complementarity between the siRNA and the target, butthe correspondence must be sufficient to enable the siRNA, or a cleavageproduct thereof, to direct sequence specific silencing, such as by RNAicleavage of the target RNA. Complementarity, or degree of homology withthe target strand, is most critical in the antisense strand. Whileperfect complementarity, particularly in the antisense strand, is oftendesired, some embodiments include one or more, but preferably 10, 8, 6,5, 4, 3, 2, or fewer mismatches with respect to the target RNA. Themismatches are most tolerated in the terminal regions, and if presentare preferably in a terminal region or regions, e.g., within 6, 5, 4, or3 nucleotides of the 5′ and/or 3′ terminus. The sense strand need onlybe sufficiently complementary with the antisense strand to maintain theoverall double-strand character of the molecule.

In addition, an siRNA may be modified or include nucleoside analogs.Single stranded regions of an siRNA may be modified or includenucleoside analogs, e.g., the unpaired region or regions of a hairpinstructure, e.g., a region which links two complementary regions, canhave modifications or nucleoside analogs. Modification to stabilize oneor more 3′- or 5′-terminus of an siRNA, e.g., against exonucleases, orto favor the antisense siRNA agent to enter into RISC are also useful.Modifications can include C3 (or C6, C7, C12) amino linkers, thiollinkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12,abasic, triethylene glycol, hexaethylene glycol), special biotin orfluorescein reagents that come as phosphoramidites and that have anotherDMT-protected hydroxyl group, allowing multiple couplings during RNAsynthesis. Each strand of an siRNA can be equal to or less than 30, 25,24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably atleast 19 nucleotides in length. For example, each strand can be between21 and 25 nucleotides in length. Preferred siRNAs have a duplex regionof 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one ormore overhangs of 2-3 nucleotides, preferably one or two 3′ overhangs,of 2-3 nucleotides.

As used herein, the terms “miRNA” or “microRNA” s refer to smallnon-coding RNAs of 20-22 nucleotides, typically excised from ˜70nucleotide foldback RNA precursor structures known as pre-miRNAs. miRNAsnegatively regulate their targets in one of two ways depending on thedegree of complementarity between the miRNA and the target. First,miRNAs that bind with perfect or nearly perfect complementarity toprotein-coding mRNA sequences induce the RNA-mediated interference(RNAi) pathway. miRNAs that exert their regulatory effects by binding toimperfect complementary sites within the 3′ untranslated regions (UTRs)of their mRNA targets, repress target-gene expressionpost-transcriptionally, apparently at the level of translation, througha RISC complex that is similar to, or possibly identical with, the onethat is used for the RNAi pathway. Consistent with translationalcontrol, miRNAs that use this mechanism reduce the protein levels oftheir target genes, but the mRNA levels of these genes are onlyminimally affected. miRNAs encompass both naturally occurring miRNAs aswell as artificially designed miRNAs that can specifically target anymRNA sequence. For example, in one embodiment, the skilled artisan candesign short hairpin RNA constructs expressed as human miRNA (e.g.,miR-30 or miR-21) primary transcripts. This design adds a Droshaprocessing site to the hairpin construct and has been shown to greatlyincrease knockdown efficiency (Pusch et al., 2004). The hairpin stemconsists of 22-nt of dsRNA (e.g., antisense has perfect complementarityto desired target) and a 15-19-nt loop from a human miR. Adding the miRloop and miR30 flanking sequences on either or both sides of the hairpinresults in greater than 10-fold increase in Drosha and Dicer processingof the expressed hairpins when compared with conventional shRNA designswithout microRNA. Increased Drosha and Dicer processing translates intogreater siRNA/miRNA production and greater potency for expressedhairpins.

As used herein, the terms “shRNA” or “short hairpin RNA” refer todouble-stranded structure that is formed by a single self-complementaryRNA strand. shRNA constructs containing a nucleotide sequence identicalto a portion, of either coding or non-coding sequence, of the targetgene are preferred for inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Greater than 90%sequence identity, or even 100% sequence identity, between theinhibitory RNA and the portion of the target gene is preferred. Incertain preferred embodiments, the length of the duplex-forming portionof an shRNA is at least 20, 21 or 22 nucleotides in length, e.g.,corresponding in size to RNA products produced by Dicer-dependentcleavage. In certain embodiments, the shRNA construct is at least 25,50, 100, 200, 300 or 400 bases in length. In certain embodiments, theshRNA construct is 400-800 bases in length. shRNA constructs are highlytolerant of variation in loop sequence and loop size.\

As used herein, the term “ribozyme” refers to a catalytically active RNAmolecule capable of site-specific cleavage of target mRNA. Severalsubtypes have been described, e.g., hammerhead and hairpin ribozymes.Ribozyme catalytic activity and stability can be improved bysubstituting deoxyribonucleotides for ribonucleotides at noncatalyticbases. While ribozymes that cleave mRNA at site-specific recognitionsequences can be used to destroy particular mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA has thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art.

A preferred method of delivery of a polynucleotide-of-interest thatcomprises an siRNA, an miRNA, an shRNA, or a ribozyme comprises one ormore regulatory sequences, such as, for example, a strong constitutivepol III, e.g., human U6 snRNA promoter, the mouse U6 snRNA promoter, thehuman and mouse H1 RNA promoter and the human tRNA-val promoter, or astrong constitutive pol II promoter, as described elsewhere herein.

The polynucleotides contemplated herein, regardless of the length of thecoding sequence itself, may be combined with other DNA sequences, suchas promoters and/or enhancers, untranslated regions (UTRs), signalsequences, Kozak sequences, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, internal ribosomalentry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, andAtt sites), termination codons, transcriptional termination signals, andpolynucleotides encoding self-cleaving polypeptides, epitope tags, asdisclosed elsewhere herein or as known in the art, such that theiroverall length may vary considerably. It is therefore contemplated thata polynucleotide fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

Polynucleotides can be prepared, manipulated and/or expressed using anyof a variety of well-established techniques known and available in theart. In order to express a desired polypeptide, a nucleotide sequenceencoding the polypeptide, can be inserted into appropriate vector.

Examples of vectors are plasmid, autonomously replicating sequences, andtransposable elements. Additional exemplary vectors include, withoutlimitation, plasmids, phagemids, cosmids, transposons, artificialchromosomes such as yeast artificial chromosome (YAC), bacterialartificial chromosome (BAC), or P1-derived artificial chromosome (PAC),bacteriophages such as lambda phage or M13 phage, and animal viruses.Examples of categories of animal viruses useful as vectors include,without limitation, retrovirus (including lentivirus), adenovirus,adeno-associated virus, herpesvirus (e.g., herpes simplex virus),poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).Examples of expression vectors are pClneo vectors (Promega) forexpression in mammalian cells; pLenti4N5-DEST™, pLenti6/V5-DEST™, andpLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transferand expression in mammalian cells. In particular embodiments, the codingsequences of the chimeric proteins disclosed herein can be ligated intosuch expression vectors for the expression of the chimeric protein inmammalian cells.

In particular embodiments, the vector is a non-integrating vector,including but not limited to, an episomal vector or a vector that ismaintained extrachromosomally. As used herein, the term “episomal”refers to a vector that is able to replicate without integration intohost's chromosomal DNA and without gradual loss from a dividing hostcell also meaning that said vector replicates extrachromosomally orepisomally. The vector is engineered to harbor the sequence coding forthe origin of DNA replication or “ori” from a lymphotrophic herpes virusor a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus,or a yeast, specifically a replication origin of a lymphotrophic herpesvirus or a gamma herpesvirus corresponding to oriP of EBV. In aparticular embodiment, the lymphotrophic herpes virus may be EpsteinBarr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes virussaimiri (HS), or Marek's disease virus (MDV). Epstein Barr virus (EBV)and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gammaherpesvirus. Typically, the host cell comprises the viral replicationtransactivator protein that activates the replication.

In particular embodiments, a polynucleotide is introduced into a targetor host cell using a transposon vector system. In certain embodiments,the transposon vector system comprises a vector comprising transposableelements and a polynucleotide contemplated herein; and a transposase. Inone embodiment, the transposon vector system is a single transposasevector system, see, e.g., International Application No. PCT/US07/18922.Exemplary transposases include, but are not limited to: piggyBac,Sleeping Beauty, Mos1, Tc1/mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I,Frog Prince, and derivatives thereof. The piggyBac transposon andtransposase are described, for example, in U.S. Pat. No. 6,962,810,which is incorporated herein by reference in its entirety. The SleepingBeauty transposon and transposase are described, for example, in Izsvaket al., J. Mol. Biol. 302: 93-102 (2000), which is incorporated hereinby reference in its entirety. The Tol2 transposon which was firstisolated from the medaka fish Oryzias latipes and belongs to the hATfamily of transposons is described in Kawakami et al. (2000). Mini-Tol2is a variant of Tol2 and is described in Balciunas et al. (2006). TheTol2 and Mini-Tol2 transposons facilitate integration of a transgeneinto the genome of an organism when co-acting with the Tol2 transposase.The Frog Prince transposon and transposase are described, for example,in Miskey et al., Nucleic Acids Res. 31:6873-6881 (2003).

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of the vector—originof replication, selection cassettes, promoters, enhancers, translationinitiation signals (Shine Dalgarno sequence or Kozak sequence) introns,a polyadenylation sequence, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including ubiquitous promotersand inducible promoters may be used.

In particular embodiments, vectors include, but not limited toexpression vectors and viral vectors, will include exogenous,endogenous, or heterologous control sequences such as promoters and/orenhancers. An “endogenous” control sequence is one which is naturallylinked with a given gene in the genome. An “exogenous” control sequenceis one which is placed in juxtaposition to a gene by means of geneticmanipulation (i.e., molecular biological techniques) such thattranscription of that gene is directed by the linked enhancer/promoter.A “heterologous” control sequence is an exogenous sequence that is froma different species than the cell being genetically manipulated.

The term “promoter” as used herein refers to a recognition site of apolynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNApolymerase initiates and transcribes polynucleotides operably linked tothe promoter. In particular embodiments, promoters operative inmammalian cells comprise an AT-rich region located approximately 25 to30 bases upstream from the site where transcription is initiated and/oranother sequence found 70 to 80 bases upstream from the start oftranscription, a CNCAAT region where N may be any nucleotide.

The term “enhancer” refers to a segment of DNA which contains sequencescapable of providing enhanced transcription and in some instances canfunction independent of their orientation relative to another controlsequence. An enhancer can function cooperatively or additively withpromoters and/or other enhancer elements. The term “promoter/enhancer”refers to a segment of DNA which contains sequences capable of providingboth promoter and enhancer functions.

The term “operably linked”, refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. In one embodiment, the term refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, and/or enhancer) and a second polynucleotidesequence, e.g., a polynucleotide-of-interest, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

As used herein, the term “constitutive expression control sequence”refers to a promoter, enhancer, or promoter/enhancer that continually orcontinuously allows for transcription of an operably linked sequence. Aconstitutive expression control sequence may be a “ubiquitous” promoter,enhancer, or promoter/enhancer that allows expression in a wide varietyof cell and tissue types or a “cell specific,” “cell type specific,”“cell lineage specific,” or “tissue specific” promoter, enhancer, orpromoter/enhancer that allows expression in a restricted variety of celland tissue types, respectively.

Illustrative ubiquitous expression control sequences suitable for use inparticular embodiments include, but are not limited to, acytomegalovirus (CMV) immediate early promoter, a viral simian virus 40(SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV)LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus(HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters fromvaccinia virus, an elongation factor 1-alpha (EF1a) promoter, earlygrowth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL),Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translationinitiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5),heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein70 kDa (HSP70), β-kinesin (β-KIN), the human ROSA 26 locus (Irions etal., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C promoter(UBC), a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirusenhancer/chicken β-actin (CAG) promoter, a β-actin promoter and amyeloproliferative sarcoma virus enhancer, negative control regiondeleted, d1587rev primer-binding site substituted (MND) promoter(Challita et al., J Virol. 69(2):748-55 (1995)).

In one embodiment, a vector comprises an MND promoter.

In one embodiment, a vector comprises an EF1a promoter comprising thefirst intron of the human EF1a gene.

In one embodiment, a vector comprises an EF1a promoter that lacks thefirst intron of the human EF1a gene.

In a particular embodiment, it may be desirable to express apolynucleotide comprising a CAR from a T cell specific promoter.

As used herein, “conditional expression” may refer to any type ofconditional expression including, but not limited to, inducibleexpression; repressible expression; expression in cells or tissueshaving a particular physiological, biological, or disease state, etc.This definition is not intended to exclude cell type or tissue specificexpression. Certain embodiments provide conditional expression of apolynucleotide-of-interest, e.g., expression is controlled by subjectinga cell, tissue, organism, etc., to a treatment or condition that causesthe polynucleotide to be expressed or that causes an increase ordecrease in expression of the polynucleotide encoded by thepolynucleotide-of-interest.

Illustrative examples of inducible promoters/systems include, but arenot limited to, steroid-inducible promoters such as promoters for genesencoding glucocorticoid or estrogen receptors (inducible by treatmentwith the corresponding hormone), metallothionine promoter (inducible bytreatment with various heavy metals), MX-1 promoter (inducible byinterferon), the “GeneSwitch” mifepristone-regulatable system (Sirin etal., 2003, Gene, 323:67), the cumate inducible gene switch (WO2002/088346), tetracycline-dependent regulatory systems, etc.

Conditional expression can also be achieved by using a site specific DNArecombinase. According to certain embodiments the vector comprises atleast one (typically two) site(s) for recombination mediated by a sitespecific recombinase. As used herein, the terms “recombinase” or “sitespecific recombinase” include excisive or integrative proteins, enzymes,co-factors or associated proteins that are involved in recombinationreactions involving one or more recombination sites (e.g., two, three,four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.),which may be wild-type proteins (see Landy, Current Opinion inBiotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusionproteins containing the recombination protein sequences or fragmentsthereof), fragments, and variants thereof. Illustrative examples ofrecombinases suitable for use in particular embodiments include, but arenot limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

The vectors may comprise one or more recombination sites for any of awide variety of site specific recombinases. It is to be understood thatthe target site for a site specific recombinase is in addition to anysite(s) required for integration of a vector, e.g., a retroviral vectoror lentiviral vector. As used herein, the terms “recombinationsequence,” “recombination site,” or “site specific recombination site”refer to a particular nucleic acid sequence to which a recombinaserecognizes and binds.

For example, one recombination site for Cre recombinase is loxP which isa 34 base pair sequence comprising two 13 base pair inverted repeats(serving as the recombinase binding sites) flanking an 8 base pair coresequence (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology5:521-527 (1994)). Other exemplary loxP sites include, but are notlimited to: lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171(Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al.,2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

Suitable recognition sites for the FLP recombinase include, but are notlimited to: FRT (McLeod, et al., 1996), F₁, F₂, F₃ (Schlake and Bode,1994), F₄, F₅ (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988),FRT(RE) (Senecoff et al., 1988).

Other examples of recognition sequences are the attB, attP, attL, andattR sequences, which are recognized by the recombinase enzyme λIntegrase, e.g., phi-c31. The φC31 SSR mediates recombination onlybetween the heterotypic sites attB (34 bp in length) and attP (39 bp inlength) (Groth et al., 2000). attB and attP, named for the attachmentsites for the phage integrase on the bacterial and phage genomes,respectively, both contain imperfect inverted repeats that are likelybound by φC31 homodimers (Groth et al., 2000). The product sites, attLand attR, are effectively inert to further φC31-mediated recombination(Belteki et al., 2003), making the reaction irreversible. For catalyzinginsertions, it has been found that attB-bearing DNA inserts into agenomic attP site more readily than an attP site into a genomic attBsite (Thyagaraj an et al., 2001; Belteki et al., 2003). Thus, typicalstrategies position by homologous recombination an attP-bearing “dockingsite” into a defined locus, which is then partnered with an attB-bearingincoming sequence for insertion.

As used herein, an “internal ribosome entry site” or “IRES” refers to anelement that promotes direct internal ribosome entry to the initiationcodon, such as ATG, of a cistron (a protein encoding region), therebyleading to the cap-independent translation of the gene. See, e.g.,Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson andKaminski. 1995. RNA 1(10):985-1000. In particular embodiments, vectorsinclude one or more polynucleotides-of-interest that encode one or morepolypeptides. In particular embodiments, to achieve efficienttranslation of each of the plurality of polypeptides, the polynucleotidesequences can be separated by one or more IRES sequences orpolynucleotide sequences encoding self-cleaving polypeptides.

As used herein, the term “Kozak sequence” refers to a short nucleotidesequence that greatly facilitates the initial binding of mRNA to thesmall subunit of the ribosome and increases translation. The consensusKozak sequence is (GCC)RCCATGG (SEQ ID NO:57), where R is a purine (A orG) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res.15(20):8125-48). In particular embodiments, the vectors comprisepolynucleotides that have a consensus Kozak sequence and that encode adesired polypeptide, e.g., a CAR.

In some embodiments, a polynucleotide or cell harboring thepolynucleotide utilizes a suicide gene, including an inducible suicidegene to reduce the risk of direct toxicity and/or uncontrolledproliferation. In specific embodiments, the suicide gene is notimmunogenic to the host harboring the polynucleotide or cell. A certainexample of a suicide gene that may be used is caspase-9 or caspase-8 orcytosine deaminase. Caspase-9 can be activated using a specific chemicalinducer of dimerization (CID).

In certain embodiments, vectors comprise gene segments that cause theimmune effector cells, e.g., T cells, to be susceptible to negativeselection in vivo. By “negative selection” is meant that the infusedcell can be eliminated as a result of a change in the in vivo conditionof the individual. The negative selectable phenotype may result from theinsertion of a gene that confers sensitivity to an administered agent,for example, a compound. Negative selectable genes are known in the art,and include, inter alia the following: the Herpes simplex virus type Ithymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11:223, 1977)which confers ganciclovir sensitivity; the cellular hypoxanthinephosphribosyltransferase (HPRT) gene, the cellular adeninephosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase,(Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some embodiments, genetically modified immune effector cells, such asT cells, comprise a polynucleotide further comprising a positive markerthat enables the selection of cells of the negative selectable phenotypein vitro. The positive selectable marker may be a gene which, upon beingintroduced into the host cell expresses a dominant phenotype permittingpositive selection of cells carrying the gene. Genes of this type areknown in the art, and include, inter alia, hygromycin-Bphosphotransferase gene (hph) which confers resistance to hygromycin B,the amino glycoside phosphotransferase gene (neo or aph) from Tn5 whichcodes for resistance to the antibiotic G418, the dihydrofolate reductase(DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drugresistance (MDR) gene.

Preferably, the positive selectable marker and the negative selectableelement are linked such that loss of the negative selectable elementnecessarily also is accompanied by loss of the positive selectablemarker. Even more preferably, the positive and negative selectablemarkers are fused so that loss of one obligatorily leads to loss of theother. An example of a fused polynucleotide that yields as an expressionproduct a polypeptide that confers both the desired positive andnegative selection features described above is a hygromycinphosphotransferase thymidine kinase fusion gene (HyTK). Expression ofthis gene yields a polypeptide that confers hygromycin B resistance forpositive selection in vitro, and ganciclovir sensitivity for negativeselection in vivo. See Lupton S. D., et al, Mol. and Cell. Biology11:3374-3378, 1991. In addition, in preferred embodiments, thepolynucleotides encoding the chimeric receptors are in retroviralvectors containing the fused gene, particularly those that conferhygromycin B resistance for positive selection in vitro, and ganciclovirsensitivity for negative selection in vivo, for example the HyTKretroviral vector described in Lupton, S. D. et al. (1991), supra. Seealso the publications of PCT US91/08442 and PCT/US94/05601, by S. D.Lupton, describing the use of bifunctional selectable fusion genesderived from fusing a dominant positive selectable markers with negativeselectable markers.

Preferred positive selectable markers are derived from genes selectedfrom the group consisting of hph, nco, and gpt, and preferred negativeselectable markers are derived from genes selected from the groupconsisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt.Especially preferred markers are bifunctional selectable fusion geneswherein the positive selectable marker is derived from hph or neo, andthe negative selectable marker is derived from cytosine deaminase or aTK gene or selectable marker.

In various embodiments, the polynucleotide is an mRNA that is introducedinto a cell in order to transiently express a desired polypeptide. Asused herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thepolynucleotide if integrated into the genome or contained within astable vector or plasmid in the cell.

In particular embodiments, the mRNA encoding a polypeptide is an invitro transcribed mRNA. As used herein, “in vitro transcribed RNA”refers to RNA, preferably mRNA, that has been synthesized in vitro.Generally, the in vitro transcribed RNA is generated from an in vitrotranscription vector. The in vitro transcription vector comprises atemplate that is used to generate the in vitro transcribed RNA.

In particular embodiments, mRNAs further comprise a comprise a 5′ cap ormodified 5′ cap and/or a poly(A) sequence. As used herein, a 5′ cap(also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m^(7G)cap) is a modified guanine nucleotide that has been added to the “front”or 5′ end of a eukaryotic messenger RNA shortly after the start oftranscription. The 5′ cap comprises a terminal group which is linked tothe first transcribed nucleotide and recognized by the ribosome andprotected from RNases. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation. In a particular embodiment, the mRNA comprises a poly(A)sequence of between about 50 and about 5000 adenines. In one embodiment,the mRNA comprises a poly(A) sequence of between about 100 and about1000 bases, between about 200 and about 500 bases, or between about 300and about 400 bases. In one embodiment, the mRNA comprises a poly(A)sequence of about 65 bases, about 100 bases, about 200 bases, about 300bases, about 400 bases, about 500 bases, about 600 bases, about 700bases, about 800 bases, about 900 bases, or about 1000 or more bases.poly(A) sequences can be modified chemically or enzymatically tomodulate mRNA functionality such as localization, stability orefficiency of translation.

F. Viral Vectors

In particular embodiments, a cell (e.g., an immune effector cell) istransduced with a retroviral vector, e.g., a lentiviral vector, encodinga CAR. For example, an immune effector cell is transduced with a vectorencoding a CAR that comprises a an anti-STn antibody or antigen bindingfragment thereof that binds an STn expressing glycoprotein, e.g.,TAG-72, with an intracellular signaling domain of CD3ζ, CD28, 4-1BB,OX40, or any combinations thereof. Thus, these transduced cells canelicit a CAR-mediated cytotoxic response.

Retroviruses are a common tool for gene delivery (Miller, 2000, Nature.357: 455-460). In particular embodiments, a retrovirus is used todeliver a polynucleotide encoding a chimeric antigen receptor (CAR) to acell. As used herein, the term “retrovirus” refers to an RNA virus thatreverse transcribes its genomic RNA into a linear double-stranded DNAcopy and subsequently covalently integrates its genomic DNA into a hostgenome. Once the virus is integrated into the host genome, it isreferred to as a “provirus.” The provirus serves as a template for RNApolymerase II and directs the expression of RNA molecules which encodethe structural proteins and enzymes needed to produce new viralparticles.

Illustrative retroviruses suitable for use in particular embodiments,include, but are not limited to: Moloney murine leukemia virus (M-MuLV),Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus(GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemiavirus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) andlentivirus.

As used herein, the term “lentivirus” refers to a group (or genus) ofcomplex retroviruses. Illustrative lentiviruses include, but are notlimited to: HIV (human immunodeficiency virus; including HIV type 1, andHIV type 2); visna-maedi virus (VMV) virus; the caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (FIV); bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV). In one embodiment,HIV based vector backbones (i.e., HIV cis-acting sequence elements) arepreferred. In particular embodiments, a lentivirus is used to deliver apolynucleotide comprising a CAR to a cell.

Retroviral vectors and more particularly lentiviral vectors may be usedin practicing particular embodiments. Accordingly, the term “retrovirus”or “retroviral vector”, as used herein is meant to include “lentivirus”and “lentiviral vectors” respectively.

The term “vector” is used herein to refer to a nucleic acid moleculecapable transferring or transporting another nucleic acid molecule. Thetransferred nucleic acid is generally linked to, e.g., inserted into,the vector nucleic acid molecule. A vector may include sequences thatdirect autonomous replication in a cell, or may include sequencessufficient to allow integration into host cell DNA. Useful vectorsinclude, for example, plasmids (e.g., DNA plasmids or RNA plasmids),transposons, cosmids, bacterial artificial chromosomes, and viralvectors. Useful viral vectors include, e.g., replication defectiveretroviruses and lentiviruses.

As will be evident to one of skill in the art, the term “viral vector”is widely used to refer either to a nucleic acid molecule (e.g., atransfer plasmid) that includes virus-derived nucleic acid elements thattypically facilitate transfer of the nucleic acid molecule orintegration into the genome of a cell or to a viral particle thatmediates nucleic acid transfer. Viral particles will typically includevarious viral components and sometimes also host cell components inaddition to nucleic acid(s).

The term viral vector may refer either to a virus or viral particlecapable of transferring a nucleic acid into a cell or to the transferrednucleic acid itself. Viral vectors and transfer plasmids containstructural and/or functional genetic elements that are primarily derivedfrom a virus. The term “retroviral vector” refers to a viral vector orplasmid containing structural and functional genetic elements, orportions thereof, that are primarily derived from a retrovirus. The term“lentiviral vector” refers to a viral vector or plasmid containingstructural and functional genetic elements, or portions thereof,including LTRs that are primarily derived from a lentivirus. The term“hybrid vector” refers to a vector, LTR or other nucleic acid containingboth retroviral, e.g., lentiviral, sequences and non-lentiviral viralsequences. In one embodiment, a hybrid vector refers to a vector ortransfer plasmid comprising retroviral e.g., lentiviral, sequences forreverse transcription, replication, integration and/or packaging.

In particular embodiments, the terms “lentiviral vector,” “lentiviralexpression vector” may be used to refer to lentiviral transfer plasmidsand/or infectious lentiviral particles. Where reference is made hereinto elements such as cloning sites, promoters, regulatory elements,heterologous nucleic acids, etc., it is to be understood that thesequences of these elements are present in RNA form in the lentiviralparticles and are present in DNA form in the DNA plasmids.

At each end of the provirus are structures called “long terminalrepeats” or “LTRs.” The term “long terminal repeat (LTR)” refers todomains of base pairs located at the ends of retroviral DNAs which, intheir natural sequence context, are direct repeats and contain U3, R andU5 regions. LTRs generally provide functions fundamental to theexpression of retroviral genes (e.g., promotion, initiation andpolyadenylation of gene transcripts) and to viral replication. The LTRcontains numerous regulatory signals including transcriptional controlelements, polyadenylation signals and sequences needed for replicationand integration of the viral genome. The viral LTR is divided into threeregions called U3, R and U5. The U3 region contains the enhancer andpromoter elements. The U5 region is the sequence between the primerbinding site and the R region and contains the polyadenylation sequence.The R (repeat) region is flanked by the U3 and U5 regions. The LTRcomposed of U3, R and U5 regions and appears at both the 5′ and 3′ endsof the viral genome. Adjacent to the 5′ LTR are sequences necessary forreverse transcription of the genome (the tRNA primer binding site) andfor efficient packaging of viral RNA into particles (the Psi site).

As used herein, the term “packaging signal” or “packaging sequence”refers to sequences located within the retroviral genome which arerequired for insertion of the viral RNA into the viral capsid orparticle, see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4;pp. 2101-2109. Several retroviral vectors use the minimal packagingsignal (also referred to as the psi [Ψ] sequence) needed forencapsidation of the viral genome. Thus, as used herein, the terms“packaging sequence,” “packaging signal,” “psi” and the symbol “Ψ,” areused in reference to the non-coding sequence required for encapsidationof retroviral RNA strands during viral particle formation.

In various embodiments, vectors comprise modified 5′ LTR and/or 3′ LTRs.Either or both of the LTR may comprise one or more modificationsincluding, but not limited to, one or more deletions, insertions, orsubstitutions. Modifications of the 3′ LTR are often made to improve thesafety of lentiviral or retroviral systems by rendering virusesreplication-defective. As used herein, the term “replication-defective”refers to virus that is not capable of complete, effective replicationsuch that infective virions are not produced (e.g.,replication-defective lentiviral progeny). The term“replication-competent” refers to wild-type virus or mutant virus thatis capable of replication, such that viral replication of the virus iscapable of producing infective virions (e.g., replication-competentlentiviral progeny).

“Self-inactivating” (SIN) vectors refers to replication-defectivevectors, e.g., retroviral or lentiviral vectors, in which the right (3′)LTR enhancer-promoter region, known as the U3 region, has been modified(e.g., by deletion or substitution) to prevent viral transcriptionbeyond the first round of viral replication. This is because the right(3′) LTR U3 region is used as a template for the left (5′) LTR U3 regionduring viral replication and, thus, the viral transcript cannot be madewithout the U3 enhancer-promoter. In a further embodiment, the 3′ LTR ismodified such that the U5 region is replaced, for example, with an idealpoly(A) sequence. It should be noted that modifications to the LTRs suchas modifications to the 3′ LTR, the 5′ LTR, or both 3′ and 5′ LTRs, arealso included.

An additional safety enhancement is provided by replacing the U3 regionof the 5′ LTR with a heterologous promoter to drive transcription of theviral genome during production of viral particles. Examples ofheterologous promoters which can be used include, for example, viralsimian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV)(e.g., immediate early), Moloney murine leukemia virus (MoMLV), Roussarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase)promoters. Typical promoters are able to drive high levels oftranscription in a Tat-independent manner. This replacement reduces thepossibility of recombination to generate replication-competent virusbecause there is no complete U3 sequence in the virus production system.In certain embodiments, the heterologous promoter has additionaladvantages in controlling the manner in which the viral genome istranscribed. For example, the heterologous promoter can be inducible,such that transcription of all or part of the viral genome will occuronly when the induction factors are present. Induction factors include,but are not limited to, one or more chemical compounds or thephysiological conditions such as temperature or pH, in which the hostcells are cultured.

In some embodiments, viral vectors comprise a TAR element. The term“TAR” refers to the “trans-activation response” genetic element locatedin the R region of lentiviral (e.g., HIV) LTRs. This element interactswith the lentiviral trans-activator (tat) genetic element to enhanceviral replication. However, this element is not required in embodimentswherein the U3 region of the 5′ LTR is replaced by a heterologouspromoter.

The “R region” refers to the region within retroviral LTRs beginning atthe start of the capping group (i.e., the start of transcription) andending immediately prior to the start of the poly(A) tract. The R regionis also defined as being flanked by the U3 and U5 regions. The R regionplays a role during reverse transcription in permitting the transfer ofnascent DNA from one end of the genome to the other.

As used herein, the term “FLAP element” refers to a nucleic acid whosesequence includes the central polypurine tract and central terminationsequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. In someembodiments, the terms “FLAP element” and “cPPT/FLAP” are usedinterchangeably to refer to the foregoing FLAP element. Suitable FLAPelements are described in U.S. Pat. No. 6,682,907 and in Zennou, et al.,2000, Cell, 101:173. During HIV-1 reverse transcription, centralinitiation of the plus-strand DNA at the central polypurine tract (cPPT)and central termination at the central termination sequence (CTS) leadto the formation of a three-stranded DNA structure: the HIV-1 centralDNA FLAP. While not wishing to be bound by any theory, the DNA FLAP mayact as a cis-active determinant of lentiviral genome nuclear importand/or may increase the titer of the virus. In particular embodiments,the retroviral or lentiviral vector backbones comprise one or more FLAPelements upstream or downstream of the heterologous genes of interest inthe vectors. For example, in particular embodiments a transfer plasmidincludes a FLAP element. In one embodiment, a vector comprises a FLAPelement isolated from HIV-1.

In one embodiment, retroviral or lentiviral transfer vectors compriseone or more export elements. The term “export element” refers to acis-acting post-transcriptional regulatory element which regulates thetransport of an RNA transcript from the nucleus to the cytoplasm of acell. Examples of RNA export elements include, but are not limited to,the human immunodeficiency virus (HIV) rev response element (RRE) (seee.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991.Cell 58: 423), and the hepatitis B virus post-transcriptional regulatoryelement (HPRE). Generally, the RNA export element is placed within the3′ UTR of a gene, and can be inserted as one or multiple copies.

In particular embodiments, expression of heterologous sequences in viralvectors is increased by incorporating posttranscriptional regulatoryelements, efficient polyadenylation sites, and optionally, transcriptiontermination signals into the vectors. A variety of posttranscriptionalregulatory elements can increase expression of a heterologous nucleicacid at the protein, e.g., woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886);the posttranscriptional regulatory element present in hepatitis B virus(HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu etal., 1995, Genes Dev., 9:1766). In particular embodiments, vectorscomprise a posttranscriptional regulatory element such as a WPRE orHPRE.

In particular embodiments, vectors lack or do not comprise aposttranscriptional regulatory element such as a WPRE or HPRE because insome instances these elements increase the risk of cellulartransformation and/or do not substantially or significantly increase theamount of mRNA transcript or increase mRNA stability. However, theeffect of including a WPRE in a given vector may be unpredictable insome embodiments. Therefore, in some embodiments, vectors lack or do notcomprise a WPRE or HPRE.

Elements directing the efficient termination and polyadenylation of theheterologous nucleic acid transcripts increases heterologous geneexpression. Transcription termination signals are generally founddownstream of the polyadenylation signal.

In particular embodiments, vectors comprise a polyadenylation sequence3′ of a polynucleotide encoding a polypeptide to be expressed. The term“poly(A) site” or “poly(A) sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript by RNA polymerase II. Polyadenylation sequencescan promote mRNA stability by addition of a poly(A) tail to the 3′ endof the coding sequence and thus, contribute to increased translationalefficiency. Cleavage and polyadenylation is directed by a poly(A)sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAshas two recognition elements flanking a cleavage-polyadenylation site.Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotidesupstream of a more variable element rich in U or GU residues. Cleavageof the nascent transcript occurs between these two elements and iscoupled to the addition of up to 250 adenosines to the 5′ cleavageproduct. In particular embodiments, the core poly(A) sequence is anideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In particularembodiments the poly(A) sequence is an SV40 poly(A) sequence, a bovinegrowth hormone poly(A) sequence (BGHpA), a rabbit β-globin poly(A)sequence (rβgpA), or another suitable heterologous or endogenous poly(A)sequence known in the art.

In certain embodiments, a retroviral or lentiviral vector furthercomprises one or more insulator elements. Insulators elements maycontribute to protecting lentivirus-expressed sequences, e.g.,therapeutic polypeptides, from integration site effects, which may bemediated by cis-acting elements present in genomic DNA and lead toderegulated expression of transferred sequences (i.e., position effect;see, e.g., Burgess-Beusse et al., 2002, Proc. Natl. Acad. Sci., USA,99:16433; and Zhan et al., 2001, Hum. Genet., 109:471). In someembodiments, transfer vectors comprise one or more insulator element the3′ LTR and upon integration of the provirus into the host genome, theprovirus comprises the one or more insulators at both the 5′ LTR or 3′LTR, by virtue of duplicating the 3′ LTR. Suitable insulators for use inparticular embodiments include, but are not limited to, the chickenβ-globin insulator (see Chung et al., 1993. Cell 74:505; Chung et al.,1997. PNAS 94:575; and Bell et al., 1999. Cell 98:387, incorporated byreference herein). Examples of insulator elements include, but are notlimited to, an insulator from a β-globin locus, such as chicken HS4.

According to certain specific embodiments, most or all of the viralvector backbone sequences are derived from a lentivirus, e.g., HIV-1.However, it is to be understood that many different sources ofretroviral and/or lentiviral sequences can be used, or combined andnumerous substitutions and alterations in certain of the lentiviralsequences may be accommodated without impairing the ability of atransfer vector to perform the functions described herein. Moreover, avariety of lentiviral vectors are known in the art, see Naldini et al.,(1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998,U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted toproduce a viral vector or transfer plasmid.

In various embodiments, vectors comprise a promoter operably linked to apolynucleotide encoding a CAR polypeptide. The vectors may have one ormore LTRs, wherein either LTR comprises one or more modifications, suchas one or more nucleotide substitutions, additions, or deletions. Thevectors may further comprise one of more accessory elements to increasetransduction efficiency (e.g., a cPPT/FLAP), viral packaging (e.g., aPsi (T) packaging signal, RRE), and/or other elements that increasetherapeutic gene expression (e.g., poly (A) sequences), and mayoptionally comprise a WPRE or HPRE.

In a particular embodiment, a transfer vector comprises a left (5′)retroviral LTR; a central polypurine tract/DNA flap (cPPT/FLAP); aretroviral export element; a promoter active in a T cell, operablylinked to a polynucleotide encoding CAR polypeptide contemplated herein;and a right (3′) retroviral LTR; and optionally a WPRE or HPRE.

In a particular embodiment, a transfer vector comprises a left (5′)retroviral LTR; a retroviral export element; a promoter active in a Tcell, operably linked to a polynucleotide encoding CAR polypeptidecontemplated herein; a right (3′) retroviral LTR; and a poly (A)sequence; and optionally a WPRE or HPRE. In another particularembodiment, a lentiviral vector comprises: a left (5′) LTR; a cPPT/FLAP;an RRE; a promoter active in a T cell, operably linked to apolynucleotide encoding CAR polypeptide contemplated herein; a right(3′) LTR; and a polyadenylation sequence; and optionally a WPRE or HPRE.

In a certain embodiment, a lentiviral vector comprises: a left (5′)HIV-1 LTR; a Psi (T) packaging signal; a cPPT/FLAP; an RRE; a promoteractive in a T cell, operably linked to a polynucleotide encoding CARpolypeptide contemplated herein; a right (3′) self-inactivating (SIN)HIV-1 LTR; and a rabbit β-globin polyadenylation sequence; andoptionally a WPRE or HPRE.

In another embodiment, a vector comprises: at least one LTR; a centralpolypurine tract/DNA flap (cPPT/FLAP); a retroviral export element; anda promoter active in a T cell, operably linked to a polynucleotideencoding CAR polypeptide contemplated herein; and optionally a WPRE orHPRE.

In particular embodiment, a vector comprises at least one LTR; acPPT/FLAP; an RRE; a promoter active in a T cell, operably linked to apolynucleotide encoding CAR polypeptide contemplated herein; and apolyadenylation sequence; and optionally a WPRE or HPRE.

In a certain embodiment, a vector comprises at least one SIN HIV-1 LTR;a Psi (T) packaging signal; a cPPT/FLAP; an RRE; a promoter active in aT cell, operably linked to a polynucleotide encoding CAR polypeptidecontemplated herein; and a rabbit β-globin polyadenylation sequence; andoptionally a WPRE or HPRE.

A “host cell” includes cells electroporated, transfected, infected, ortransduced in vivo, ex vivo, or in vitro with a recombinant vector or apolynucleotide. Host cells may include packaging cells, producer cells,and cells infected with viral vectors. In particular embodiments, hostcells infected with viral vector are administered to a subject in needof therapy. In certain embodiments, the term “target cell” is usedinterchangeably with host cell and refers to transfected, infected, ortransduced cells of a desired cell type. In preferred embodiments, thetarget cell is a T cell.

Large scale viral particle production is often necessary to achieve areasonable viral titer. Viral particles are produced by transfecting atransfer vector into a packaging cell line that comprises viralstructural and/or accessory genes, e.g., gag, pol, env, tat, rev, vif,vpr, vpu, vpx, or nef genes or other retroviral genes.

As used herein, the term “packaging vector” refers to an expressionvector or viral vector that lacks a packaging signal and comprises apolynucleotide encoding one, two, three, four or more viral structuraland/or accessory genes. Typically, the packaging vectors are included ina packaging cell, and are introduced into the cell via transfection,transduction or infection. Methods for transfection, transduction orinfection are well known by those of skill in the art. In particularembodiments, a retroviral/lentiviral transfer vector is introduced intoa packaging cell line, via transfection, transduction or infection, togenerate a producer cell or cell line. In particular embodiments,packaging vectors are introduced into human cells or cell lines bystandard methods including, e.g., calcium phosphate transfection,lipofection or electroporation. In some embodiments, the packagingvectors are introduced into the cells together with a dominantselectable marker, such as neomycin, hygromycin, puromycin, blastocidin,zeocin, thymidine kinase, DHFR, Gln synthetase or ADA, followed byselection in the presence of the appropriate drug and isolation ofclones. A selectable marker gene can be linked physically to genesencoding by the packaging vector, e.g., by IRES or self-cleaving viralpeptides.

Viral envelope proteins (env) determine the range of host cells whichcan ultimately be infected and transformed by recombinant retrovirusesgenerated from the cell lines. In the case of lentiviruses, such asHIV-1, HIV-2, SIV, FIV and EIV, the env proteins include gp41 and gp120.Preferably, the viral env proteins expressed by packaging cells areencoded on a separate vector from the viral gag and pol genes, as hasbeen previously described.

Illustrative examples of retroviral-derived env genes which can beemployed in particular embodiments include, but are not limited to: MLVenvelopes, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV(Fowl plague virus), and influenza virus envelopes. Similarly, genesencoding envelopes from RNA viruses (e.g., RNA virus families ofPicornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae,Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae,Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae,Retroviridae) as well as from the DNA viruses (families ofHepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae,Herpesviridae, Poxyiridae, and Iridoviridae) may be utilized.Representative examples of these viruses include, but are not limitedto, FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BLV, EBV, CAEV, SNV,ChTLV, STLV, MPMV, SMRV, RAV, FuSV, AEV, AMV, CT10, and EIAV.

In other embodiments, envelope proteins for pseudotyping a virusinclude, but are not limited to any of the following virus: Influenza Asuch as H1N1, H1N2, H3N2 and H5N1 (bird flu), Influenza B, Influenza Cvirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus,Hepatitis D virus, Hepatitis E virus, Rotavirus, any virus of theNorwalk virus group, enteric adenoviruses, parvovirus, Dengue fevervirus, Monkey pox, Mononegavirales, Lyssavirus such as rabies virus,Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2and Australian bat virus, Ephemerovirus, Vesiculovirus, VesicularStomatitis Virus (VSV), Herpesviruses such as Herpes simplex virus types1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV),human herpesviruses (HHV), human herpesvirus type 6 and 8, Humanimmunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus,Arenaviruses such as Argentine hemorrhagic fever virus, Bolivianhemorrhagic fever virus, Sabia-associated hemorrhagic fever virus,Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus,Lymphocytic choriomeningitis virus (LCMV), Bunyaviridiae such asCrimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic feverwith renal syndrome causing virus, Rift Valley fever virus, Filoviridae(filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagicfever, Flaviviridae including Kaysanur Forest disease virus, Omskhemorrhagic fever virus, Tick-borne encephalitis causing virus andParamyxoviridae such as Hendra virus and Nipah virus, variola major andvariola minor (smallpox), alphaviruses such as Venezuelan equineencephalitis virus, eastern equine encephalitis virus, western equineencephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nilevirus, any encephaliltis causing virus.

In one embodiment, packaging cells produce a recombinant retrovirus,e.g., lentivirus, pseudotyped with the VSV-G glycoprotein.

The terms “pseudotype” or “pseudotyping” as used herein, refer to avirus whose viral envelope proteins have been substituted with those ofanother virus possessing preferable characteristics. For example, HIVcan be pseudotyped with vesicular stomatitis virus G-protein (VSV-G)envelope proteins, which allows HIV to infect a wider range of cellsbecause HIV envelope proteins (encoded by the env gene) normally targetthe virus to CD4+ presenting cells. In a preferred embodiment,lentiviral envelope proteins are pseudotyped with VSV-G. In oneembodiment, packaging cells produce a recombinant retrovirus, e.g.,lentivirus, pseudotyped with the VSV-G envelope glycoprotein.

As used herein, the term “packaging cell lines” is used in reference tocell lines that do not contain a packaging signal, but do stably ortransiently express viral structural proteins and replication enzymes(e.g., gag, pol and env) which are necessary for the correct packagingof viral particles. Any suitable cell line can be employed to preparepackaging cells. Generally, the cells are mammalian cells. In aparticular embodiment, the cells used to produce the packaging cell lineare human cells. Suitable cell lines which can be used include, forexample, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells,Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRCS cells,A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells,NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh? cells, HeLa cells, W163cells, 211 cells, and 211A cells. In preferred embodiments, thepackaging cells are 293 cells, 293T cells, or A549 cells. In anotherpreferred embodiment, the cells are A549 cells.

As used herein, the term “producer cell line” refers to a cell linewhich is capable of producing recombinant retroviral particles,comprising a packaging cell line and a transfer vector constructcomprising a packaging signal. The production of infectious viralparticles and viral stock solutions may be carried out usingconventional techniques. Methods of preparing viral stock solutions areknown in the art and are illustrated by, e.g., Y. Soneoka et al. (1995)Nucl. Acids Res. 23:628-633, and N. R. Landau et al. (1992) J. Virol.66:5110-5113. Infectious virus particles may be collected from thepackaging cells using conventional techniques. For example, theinfectious particles can be collected by cell lysis, or collection ofthe supernatant of the cell culture, as is known in the art. Optionally,the collected virus particles may be purified if desired. Suitablepurification techniques are well known to those skilled in the art.

The delivery of a gene(s) or other polynucleotide sequence using aretroviral or lentiviral vector by means of viral infection rather thanby transfection is referred to as “transduction.” In one embodiment,retroviral vectors are transduced into a cell through infection andprovirus integration. In certain embodiments, a target cell, e.g., a Tcell, is “transduced” if it comprises a gene or other polynucleotidesequence delivered to the cell by infection using a viral or retroviralvector. In particular embodiments, a transduced cell comprises one ormore genes or other polynucleotide sequences delivered by a retroviralor lentiviral vector in its cellular genome.

In particular embodiments, host cells transduced with viral vector thatexpresses one or more polypeptides, are administered to a subject totreat and/or prevent a B cell malignancy. Other methods relating to theuse of viral vectors in gene therapy, which may be utilized according tocertain embodiments, can be found in, e.g., Kay, M. A. (1997) Chest111(6 Supp.):138S-142S; Ferry, N. and Heard, J. M. (1998) Hum. GeneTher. 9:1975-81; Shiratory, Y. et al. (1999) Liver 19:265-74; Oka, K. etal. (2000) Curr. Opin. 11:179-86; Thule, P. M. and Liu, J. M. (2000)Gene Ther. 7:1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol.12:335-56; Alt, M. (1995) J. Hepatol. 23:746-58; Brody, S. L. andCrystal, R. G. (1994) Ann. N.Y. Acad. Sci. 716:90-101; Strayer, D. S.(1999) Expert Opin. Investig. Drugs 8:2159-2172; Smith-Arica, J. R. andBartlett, J. S. (2001) Curr. Cardiol. Rep. 3:43-49; and Lee, H. C. etal. (2000) Nature 408:483-8.

G. Genetically Modified Cells

In various embodiments, cells genetically modified to express the CARscontemplated herein, for use in the treatment of cancer are provided. Asused herein, the term “genetically engineered” or “genetically modified”refers to the addition of extra genetic material in the form of DNA orRNA into the total genetic material in a cell. The terms, “geneticallymodified cells,” “modified cells,” and, “redirected cells,” are usedinterchangeably. As used herein, the term “gene therapy” refers to theintroduction of extra genetic material in the form of DNA or RNA intothe total genetic material in a cell that restores, corrects, ormodifies expression of a gene, or for the purpose of expressing atherapeutic polypeptide, e.g., a CAR.

In particular embodiments, the CARs contemplated herein are introducedand expressed in immune effector cells so as to redirect theirspecificity to a target antigen of interest, e.g., an STn expressingglycoprotein. An “immune effector cell,” is any cell of the immunesystem that has one or more effector functions (e.g., cytotoxic cellkilling activity, secretion of cytokines, induction of ADCC and/or CDC).The illustrative immune effector cells contemplated herein are Tlymphocytes, in particular cytotoxic T cells (CTLs; CD8+ T cells), TILs,and helper T cells (HTLs; CD4+ T cells. In one embodiment, immuneeffector cells include natural killer (NK) cells. In one embodiment,immune effector cells include natural killer T (NKT) cells.

Immune effector cells can be autologous/autogeneic (“self”) ornon-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic).

“Autologous,” as used herein, refers to cells from the same subject.

“Allogeneic,” as used herein, refers to cells of the same species thatdiffer genetically to the cell in comparison.

“Syngeneic,” as used herein, refers to cells of a different subject thatare genetically identical to the cell in comparison.

“Xenogeneic,” as used herein, refers to cells of a different species tothe cell in comparison. In preferred embodiments, the cells areallogeneic.

Illustrative immune effector cells used with the CARs contemplatedherein include T lymphocytes. The terms “T cell” or “T lymphocyte” areart-recognized and are intended to include thymocytes, immature Tlymphocytes, mature T lymphocytes, resting T lymphocytes, or activated Tlymphocytes. A T cell can be a T helper (Th) cell, for example a Thelper 1 (Th1) or a T helper 2 (Th2) cell. The T cell can be a helper Tcell (HTL; CD4⁺ T cell) CD4⁺ T cell, a cytotoxic T cell (CTL; CD8⁺ Tcell), CD4⁺ CD8⁺ T cell, CD4⁻ CD8⁻ T cell, or any other subset of Tcells. Other illustrative populations of T cells suitable for use inparticular embodiments include naïve T cells and memory T cells.

As would be understood by the skilled person, other cells may also beused as immune effector cells with the CARs as described herein. Inparticular, immune effector cells also include NK cells, NKT cells,neutrophils, and macrophages. Immune effector cells also includeprogenitors of effector cells wherein such progenitor cells can beinduced to differentiate into an immune effector cells in vivo or invitro. Thus, in particular embodiments, immune effector cell includesprogenitors of immune effectors cells such as hematopoietic stem cells(HSCs) contained within the CD34⁺ population of cells derived from cordblood, bone marrow or mobilized peripheral blood which uponadministration in a subject differentiate into mature immune effectorcells, or which can be induced in vitro to differentiate into matureimmune effector cells.

As used herein, immune effector cells genetically engineered to containan STn-specific CAR may be referred to as, “STn-specific redirectedimmune effector cells.”

The term, “CD34⁺ cell,” as used herein refers to a cell expressing theCD34 protein on its cell surface. “CD34,” as used herein refers to acell surface glycoprotein (e.g., sialomucin protein) that often acts asa cell-cell adhesion factor and is involved in T cell entrance intolymph nodes. The CD34⁺ cell population contains hematopoietic stem cells(HSC), which upon administration to a patient differentiate andcontribute to all hematopoietic lineages, including T cells, NK cells,NKT cells, neutrophils and cells of the monocyte/macrophage lineage.

Methods for making the immune effector cells which express the CARcontemplated herein are provided in particular embodiments. In oneembodiment, the method comprises transfecting or transducing immuneeffector cells isolated from an individual such that the immune effectorcells express one or more CAR as described herein. In certainembodiments, the immune effector cells are isolated from an individualand genetically modified without further manipulation in vitro. Suchcells can then be directly re-administered into the individual. Infurther embodiments, the immune effector cells are first activated andstimulated to proliferate in vitro prior to being genetically modifiedto express a CAR. In this regard, the immune effector cells may becultured before and/or after being genetically modified (i.e transducedor transfected to express a CAR contemplated herein).

In particular embodiments, prior to in vitro manipulation or geneticmodification of the immune effector cells described herein, the sourceof cells is obtained from a subject. In particular embodiments, theCAR-modified immune effector cells comprise T cells.

In particular embodiments, PBMC may be directly genetically modified toexpress CARs using methods contemplated herein. In certain embodiments,after isolation of PBMC, T lymphocytes are further isolated and incertain embodiments, both cytotoxic and helper T lymphocytes can besorted into naïve, memory, and effector T cell subpopulations eitherbefore or after genetic modification and/or expansion.

The immune effector cells, such as T cells, can be genetically modifiedfollowing isolation using known methods, or the immune effector cellscan be activated and expanded (or differentiated in the case ofprogenitors) in vitro prior to being genetically modified. In aparticular embodiment, the immune effector cells, such as T cells, aregenetically modified with the chimeric antigen receptors contemplatedherein (e.g., transduced with a viral vector comprising a nucleic acidencoding a CAR) and then are activated and expanded in vitro. In variousembodiments, T cells can be activated and expanded before or aftergenetic modification to express a CAR, using methods as described, forexample, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964;5,858,358; 6,887,466; 6,905,681; 7, 144,575; 7,067,318; 7, 172,869;7,232,566; 7, 175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; andU.S. Patent Application Publication No. 20060121005.

In one embodiment, CD34⁺ cells are transduced with a nucleic acidconstruct contemplated herein. In certain embodiments, the transducedCD34⁺ cells differentiate into mature immune effector cells in vivofollowing administration into a subject, generally the subject from whomthe cells were originally isolated. In another embodiment, CD34⁺ cellsmay be stimulated in vitro prior to exposure to or after beinggenetically modified with a CAR as described herein, with one or more ofthe following cytokines: Flt-3 ligand (FLT3), stem cell factor (SCF),megakaryocyte growth and differentiation factor (TPO), IL-3 and IL-6according to the methods described previously (Asheuer et al., 2004;Imren, et al., 2004).

In particular embodiments, a population of modified immune effectorcells for the treatment of cancer comprises a CAR as disclosed herein.For example, a population of modified immune effector cells are preparedfrom peripheral blood mononuclear cells (PBMCs) obtained from a patientdiagnosed with B cell malignancy described herein (autologous donors).The PBMCs form a heterogeneous population of T lymphocytes that can beCD4⁺, CD8⁺, or CD4⁺ and CD8⁺.

The PBMCs also can include other cytotoxic lymphocytes such as NK cellsor NKT cells. An expression vector carrying the coding sequence of a CARcontemplated herein can be introduced into a population of human donor Tcells, NK cells or NKT cells. In particular embodiments, successfullytransduced T cells that carry the expression vector can be sorted usingflow cytometry to isolate CD3 positive T cells and then furtherpropagated to increase the number of these CAR protein expressing Tcells in addition to cell activation using anti-CD3 antibodies and oranti-CD28 antibodies and IL-2 or any other methods known in the art asdescribed elsewhere herein. Standard procedures are used forcryopreservation of T cells expressing the CAR protein T cells forstorage and/or preparation for use in a human subject. In oneembodiment, the in vitro transduction, culture and/or expansion of Tcells are performed in the absence of non-human animal derived productssuch as fetal calf serum and fetal bovine serum. Since a heterogeneouspopulation of PBMCs is genetically modified, the resultant transducedcells are a heterogeneous population of modified cells comprising an STnexpressing glycoprotein targeting CAR as contemplated herein.

In a further embodiment, a mixture of, e.g., one, two, three, four, fiveor more, different expression vectors can be used in geneticallymodifying a donor population of immune effector cells wherein eachvector encodes a different chimeric antigen receptor protein ascontemplated herein. The resulting modified immune effector cells formsa mixed population of modified cells, with a proportion of the modifiedcells expressing more than one different CAR proteins.

H. T Cell Manufacturing Methods

In various embodiments, genetically modified T cells are expanded bycontact with an agent that stimulates a CD3 TCR complex associatedsignal and a ligand that stimulates a co-stimulatory molecule on thesurface of the T cells.

In particular embodiments, PBMCs or isolated T cells are contacted witha stimulatory agent and costimulatory agent, such as soluble anti-CD3and anti-CD28 antibodies, or antibodies attached to a bead or othersurface, in a culture medium with appropriate cytokines, such as IL-2,IL-7, and/or IL-15.

In particular embodiments, PBMCs or isolated T cells are contacted witha stimulatory agent and costimulatory agent, such as soluble anti-CD3and anti-CD28 antibodies, or antibodies attached to a bead or othersurface, in a culture medium with appropriate cytokines, such as IL-2,IL-7, and/or IL-15 and/or one or more agents that modulate aPI3K/Akt/mTOR cell signaling pathway.

In preferred embodiments, the T cells manufactured by the methodscontemplated herein provide improved adoptive immunotherapycompositions. Without wishing to be bound to any particular theory, itis believed that the T cell compositions manufactured by the methods inparticular embodiments contemplated herein are imbued with superiorproperties, including increased survival, expansion in the relativeabsence of differentiation, and persistence in vivo. In one embodiment,a method of manufacturing T cells comprises contacting the cells withone or more agents that modulate a PI3K cell signaling pathway. In oneembodiment, a method of manufacturing T cells comprises contacting thecells with one or more agents that modulate a PI3K/Akt/mTOR cellsignaling pathway. In various embodiments, the T cells may be obtainedfrom any source and contacted with the agent during the activationand/or expansion phases of the manufacturing process. The resulting Tcell compositions are enriched in developmentally potent T cells thathave the ability to proliferate and express one or more of the followingbiomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, CD38, and CD8.In one embodiment, populations of cell comprising T cells, that havebeen treated with one or more PI3K inhibitors is enriched for apopulation of CD8⁺ T cells co-expressing one or more or, or all of, thefollowing biomarkers: CD62L, CD127, CD197, and CD38.

In one embodiment, populations of cell comprising T cells, that havebeen treated with one or more PI3K inhibitors is enriched for apopulation of CD8⁺ T cells co-expressing one or more or, or all of, thefollowing biomarkers: CD62L, CD127, CD27, and CD8.

In one embodiment, modified T cells comprising maintained levels ofproliferation and decreased differentiation are manufactured. In aparticular embodiment, T cells are manufactured by stimulating T cellsto become activated and to proliferate in the presence of one or morestimulatory signals and an agent that is an inhibitor of a PI3K cellsignaling pathway.

The T cells can then be modified to express an anti-STn CARS. In oneembodiment, the T cells are modified by transducing the T cells with aviral vector comprising an anti-STn CAR contemplated herein. In acertain embodiment, the T cells are modified prior to stimulation andactivation in the presence of an inhibitor of a PI3K cell signalingpathway. In another embodiment, T cells are modified after stimulationand activation in the presence of an inhibitor of a PI3K cell signalingpathway. In a particular embodiment, T cells are modified within 12hours, 24 hours, 36 hours, or 48 hours of stimulation and activation inthe presence of an inhibitor of a PI3K cell signaling pathway.

After T cells are activated, the cells are cultured to proliferate. Tcells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more rounds of expansion.

In various embodiments, T cell compositions are manufactured in thepresence of one or more inhibitors of a PI3K/Akt/mTOR cell signalingpathway. The inhibitors may target one or more activities in the pathwayor a single activity. Without wishing to be bound to any particulartheory, it is contemplated that treatment or contacting T cells with oneor more inhibitors of the PI3K pathway during the stimulation,activation, and/or expansion phases of the manufacturing processpreferentially increases young T cells, thereby producing superiortherapeutic T cell compositions.

In a particular embodiment, a method for increasing the proliferation ofT cells expressing an engineered T cell receptor is provided. Suchmethods may comprise, for example, harvesting a source of T cells from asubject, stimulating and activating the T cells in the presence of oneor more inhibitors of the PI3K pathway, modification of the T cells toexpress an anti-STn CAR, and expanding the T cells in culture.

In a certain embodiment, a method for producing populations of T cellsenriched for expression of one or more of the following biomarkers:CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, CD38, and CD8 iscontemplated. In one embodiment, young T cells comprise one or more of,or all of the following biological markers: CD62L, CD127, CD197, andCD38.

In one embodiment, young T cells comprise one or more of, or all of thefollowing biological markers: CD62L, CD127, CD27, and CD8.

In one embodiment, the young T cells lack expression of CD57, CD244,CD160, PD-1, CTLA4, TIM3, and LAG3 are provided. As discussed elsewhereherein, the expression levels young T cell biomarkers is relative to theexpression levels of such markers in more differentiated T cells orimmune effector cell populations.

In one embodiment, peripheral blood mononuclear cells (PBMCs) are usedas the source of T cells in the T cell manufacturing methodscontemplated herein. PBMCs form a heterogeneous population of Tlymphocytes that can be CD4⁺, CD8⁺, or CD4⁺ and CD8⁺ and can includeother mononuclear cells such as monocytes, B cells, NK cells and NKTcells. An expression vector comprising a polynucleotide encoding anengineered TCR or CAR contemplated herein can be introduced into apopulation of human donor T cells, NK cells or NKT cells. In aparticular embodiment, successfully transduced T cells that carry theexpression vector can be sorted using flow cytometry to isolate CD3positive T cells and then further propagated to increase the number ofthe modified T cells in addition to cell activation using anti-CD3antibodies and or anti-CD28 antibodies and IL-2, IL-7, and/or IL-15.

Manufacturing methods contemplated herein may further comprisecryopreservation of modified T cells for storage and/or preparation foruse in a human subject. In one embodiment, a method of storinggenetically modified murine, human or humanized CAR protein expressingimmune effector cells which target an STn expressing glycoprotein isprovided comprising cryopreserving the immune effector cells such thatthe cells remain viable upon thawing. A fraction of the immune effectorcells expressing the CAR proteins can be cryopreserved by methods knownin the art to provide a permanent source of such cells for the futuretreatment of patients afflicted with an STn expressing glycoproteinexpressed in a cancer cell. T cells are cryopreserved such that thecells remain viable upon thawing. When needed, the cryopreservedtransformed immune effector cells can be thawed, grown and expanded formore such cells. As used herein, “cryopreserving,” refers to thepreservation of cells by cooling to sub-zero temperatures, such as(typically) 77 K or −196° C. (the boiling point of liquid nitrogen).Cryoprotective agents are often used at sub-zero temperatures to preventthe cells being preserved from damage due to freezing at lowtemperatures or warming to room temperature. Cryopreservative agents andoptimal cooling rates can protect against cell injury. Cryoprotectiveagents which can be used include but are not limited to dimethylsulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395;Ashwood-Smith, Nature, 1961; 190: 1204-1205), glycerol,polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad. Sci., 1960; 85: 576), andpolyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). Thepreferred cooling rate is 1° to 3° C./minute. After at least two hours,the T cells have reached a temperature of −80° C. and can be placeddirectly into liquid nitrogen (−196° C.) for permanent storage such asin a long-term cryogenic storage vessel.

1. T Cells

The manufacture of improved CAR T cell compositions is provided inparticular embodiments. T cells used for CAR T cell production may beautologous/autogeneic (“self”) or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic). In preferred embodiments, the Tcells are obtained from a mammalian subject. In a more preferredembodiment, the T cells are obtained from a primate subject. In the mostpreferred embodiment, the T cells are obtained from a human subject.

T cells can be obtained from a number of sources including, but notlimited to, peripheral blood mononuclear cells, bone marrow, lymph nodestissue, cord blood, thymus issue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. In certainembodiments, T cells can be obtained from a unit of blood collected froma subject using any number of techniques known to the skilled person,such as sedimentation, e.g., FICOLL™ separation. In one embodiment,cells from the circulating blood of an individual are obtained byapheresis. The apheresis product typically contains lymphocytes,including T cells, monocytes, granulocytes, B cells, other nucleatedwhite blood cells, red blood cells, and platelets. In one embodiment,the cells collected by apheresis may be washed to remove the plasmafraction and to place the cells in an appropriate buffer or media forsubsequent processing. The cells can be washed with PBS or with anothersuitable solution that lacks calcium, magnesium, and most, if not allother, divalent cations. As would be appreciated by those of ordinaryskill in the art, a washing step may be accomplished by methods known tothose in the art, such as by using a semiautomated flowthroughcentrifuge. For example, the Cobe 2991 cell processor, the BaxterCytoMate, or the like. After washing, the cells may be resuspended in avariety of biocompatible buffers or other saline solution with orwithout buffer. In certain embodiments, the undesirable components ofthe apheresis sample may be removed in the cell directly resuspendedculture media.

In particular embodiments, a population of cells comprising T cells,e.g., PBMCs, is used in the manufacturing methods contemplated herein.In other embodiments, an isolated or purified population of T cells isused in the manufacturing methods contemplated herein. Cells can beisolated from peripheral blood mononuclear cells (PBMCs) by lysing thered blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL′ gradient. In some embodiments, afterisolation of PBMC, both cytotoxic and helper T lymphocytes can be sortedinto naïve, memory, and effector T cell subpopulations either before orafter activation, expansion, and/or genetic modification.

In particular embodiments, a population of cells comprising T cells,e.g., PBMCs, is used in the manufacturing methods contemplated herein.In other embodiments, an isolated or purified population of T cells isused in the manufacturing methods contemplated herein. Cells can beisolated from peripheral blood mononuclear cells (PBMCs) by lysing thered blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient. In some embodiments, afterisolation of PBMC, both cytotoxic and helper T lymphocytes can be sortedinto naïve, memory, and effector T cell subpopulations either before orafter activation, expansion, and/or genetic modification.

In particular embodiments, the population of immune effector cells ismanufactured from PBMC that are genetically modified to express CARsusing methods contemplated herein, but that are not subjected topositive or negative selection. In certain embodiments, after isolationof PBMC, T lymphocytes are further isolated and in certain embodiments,both cytotoxic and helper T lymphocytes can be sorted into naïve,memory, and effector T cell subpopulations either before or aftergenetic modification and/or expansion.

In certain embodiments, specific subpopulation of T cells, expressingone or more of the following markers: CD3, CD4, CD8, CD28, CD45RA,CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive ornegative selection techniques. In one embodiment, a specificsubpopulation of T cells, expressing one or more of the markers selectedfrom the group consisting of i) CD62L, CCR7, CD28, CD27, CD122, CD127,CD197; ii) CD62L, CD127, CD197, and CD38 or iii) CD62L, CD127, CD27, andCD8, is further isolated by positive or negative selection techniques.In various embodiments, the manufactured T cell compositions do notexpress or do not substantially express one or more of the followingmarkers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

In one embodiment, expression of one or more of the markers selectedfrom the group consisting of i) CD62L, CD127, CD197, and CD38 or ii)CD62L, CD127, CD27, and CD8, is increased at least 1.5 fold, at least 2fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10fold, at least 25 fold, or more compared to a population of T cellsactivated and expanded without a PI3K inhibitor. In one embodiment, theT cells comprise CD8⁺ T cells.

In one embodiment, expression of one or more of the markers selectedfrom the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3, andLAG3 is decreased at least 1.5 fold, at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 25 fold, ormore compared to a population of T cells activated and expanded with aPI3K inhibitor. In one embodiment, the T cells comprise CD8⁺ T cells.

In one embodiment, the manufacturing methods contemplated hereinincrease the number CART cells comprising one or more markers of naïveor developmentally potent T cells. Without wishing to be bound to anyparticular theory, the present inventors believe that treating apopulation of cells comprising T cells with one or more PI3K inhibitorsresults in an increase an expansion of developmentally potent T cellsand provides a more robust and efficacious adoptive CAR T cellimmunotherapy compared to existing CAR T cell therapies.

Illustrative examples of markers of naïve or developmentally potent Tcells increased in T cells manufactured using the methods contemplatedherein include, but are not limited to i) CD62L, CD127, CD197, and CD38or ii) CD62L, CD127, CD27, and CD8. In particular embodiments, naïve Tcells do not express do not express or do not substantially express oneor more of the following markers: CD57, CD244, CD160, PD-1, BTLA,CD45RA, CTLA4, TIM3, and LAG3.

With respect to T cells, the T cell populations resulting from thevarious expansion methodologies contemplated herein may have a varietyof specific phenotypic properties, depending on the conditions employed.In various embodiments, expanded T cell populations comprise one or moreof the following phenotypic markers: CD62L, CD27, CD127, CD197, CD38,CD8, and HLA-DR.

In one embodiment, such phenotypic markers include enhanced expressionof one or more of, or all of CD62L, CD127, CD197, and CD38. Inparticular embodiments, CD8⁺ T lymphocytes characterized by theexpression of phenotypic markers of naive T cells including CD62L,CD127, CD197, and CD38 are expanded.

In one embodiment, such phenotypic markers include enhanced expressionof one or more of, or all of CD62L, CD127, CD27, and CD8. In particularembodiments, CD8⁺ T lymphocytes characterized by the expression ofphenotypic markers of naive T cells including CD62L, CD127, CD27, andCD8 are expanded.

In particular embodiments, T cells characterized by the expression ofphenotypic markers of central memory T cells including CD45RO, CD62L,CD127, CD197, and CD38 and negative for granzyme B are expanded. In someembodiments, the central memory T cells are CD45RO⁺, CD62L⁺, CD8⁺ Tcells.

In certain embodiments, CD4⁺ T lymphocytes characterized by theexpression of phenotypic markers of naïve CD4⁺ cells including CD62L andnegative for expression of CD45RA and/or CD45RO are expanded. In someembodiments, CD4⁺ cells characterized by the expression of phenotypicmarkers of central memory CD4⁺ cells including CD62L and CD45ROpositive. In some embodiments, effector CD4⁺ cells are CD62L positiveand CD45RO negative.

In certain embodiments, the T cells are isolated from an individual andactivated and stimulated to proliferate in vitro prior to beinggenetically modified to express an anti-STn CAR. In this regard, the Tcells may be cultured before and/or after being genetically modified(i.e., transduced or transfected to express an anti-STn CAR contemplatedherein).

2. Activation and Expansion

In order to achieve sufficient therapeutic doses of T cell compositions,T cells are often subject to one or more rounds of stimulation,activation and/or expansion. T cells can be activated and expandedgenerally using methods as described, for example, in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which isincorporated herein by reference in its entirety. T cells modified toexpress an anti-STn CAR can be activated and expanded before and/orafter the T cells are modified. In addition, T cells may be contactedwith one or more agents that modulate a PI3K/Akt/mTOR cell signalingpathway before, during, and/or after activation and/or expansion. In oneembodiment, T cells manufactured by the methods contemplated hereinundergo one, two, three, four, or five or more rounds of activation andexpansion, each of which may include one or more agents that modulate aPI3K/Akt/mTOR cell signaling pathway.

Artificial antigen presenting cells (aAPCs) support ex vivo growth andlong-term expansion of functional human CD8⁺ T cells without requiringthe addition of exogenous cytokines, in contrast to the use of naturalAPCs. In particular embodiments, PBMCs or isolated T cells are contactedwith a stimulatory agent and costimulatory agent, such as anti-CD3 andanti-CD28 antibodies, generally attached to a bead or other surface, ina culture medium with appropriate cytokines, such as IL-2, IL-7, and/orIL-15.

In other embodiments, artificial APC (aAPC) made by engineering K562,U937, 721.221, T2, and C1R cells to direct the stable expression andsecretion, of a variety of costimulatory molecules and cytokines. In aparticular embodiment, K32 or U32 aAPCs are used to direct the displayof one or more antibody-based stimulatory molecules on the AAPC cellsurface. Populations of T cells can be expanded by aAPCs expressing avariety of costimulatory molecules including, but not limited to, CD137L(4-1BBL), CD134L (OX40L), and/or CD80 or CD86. The aAPCs provide anefficient platform to expand genetically modified T cells and tomaintain CD28 expression on CD8⁺ T cells. aAPCs provided in WO 03/057171and US2003/0147869 are hereby incorporated by reference in theirentirety.

In one embodiment, a costimulatory ligand is presented on an antigenpresenting cell (e.g., an aAPC, dendritic cell, B cell, and the like)that specifically binds a cognate costimulatory molecule on a T cell,thereby providing a signal which, in addition to the primary signalprovided by, for instance, binding of a TCR/CD3 complex, mediates adesired T cell response. Suitable costimulatory ligands include, but arenot limited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, induciblecostimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM),CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin betareceptor, ILT3, ILT4, an agonist or antibody that binds Toll ligandreceptor, and a ligand that specifically binds with B7-H3.

In a particular embodiment, a costimulatory ligand comprises an antibodyor antigen binding fragment thereof that specifically binds to acostimulatory molecule present on a T cell, including but not limitedto, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, and aligand that specifically binds with CD83.

Suitable costimulatory ligands further include target antigens, whichmay be provided in soluble form or expressed on APCs or aAPCs that bindengineered TCRs or CARs expressed on modified T cells.

In various embodiments, a method for manufacturing T cells contemplatedherein comprises activating a population of cells comprising T cells andexpanding the population of T cells. T cell activation can beaccomplished by providing a primary stimulation signal through the Tcell TCR/CD3 complex or via stimulation of the CD2 surface protein andby providing a secondary costimulation signal through an accessorymolecule, e.g, CD28.

The TCR/CD3 complex may be stimulated by contacting the T cell with asuitable CD3 binding agent, e.g., a CD3 ligand or an anti-CD3 monoclonalantibody. Illustrative examples of CD3 antibodies include, but are notlimited to, OKT3, G19-4, BC3, and 64.1.

In another embodiment, a CD2 binding agent may be used to provide aprimary stimulation signal to the T cells. Illustrative examples of CD2binding agents include, but are not limited to, CD2 ligands and anti-CD2antibodies, e.g., the T11.3 antibody in combination with the T11.1 orT11.2 antibody (Meuer, S. C. et al. (1984) Cell 36:897-906) and the 9.6antibody (which recognizes the same epitope as TI 1.1) in combinationwith the 9-1 antibody (Yang, S. Y. et al. (1986) J. Immunol.137:1097-1100). Other antibodies which bind to the same epitopes as anyof the above described antibodies can also be used. Additionalantibodies, or combinations of antibodies, can be prepared andidentified by standard techniques as disclosed elsewhere herein.

In addition to the primary stimulation signal provided through theTCR/CD3 complex, or via CD2, induction of T cell responses requires asecond, costimulatory signal. In particular embodiments, a CD28 bindingagent can be used to provide a costimulatory signal. Illustrativeexamples of CD28 binding agents include but are not limited to: naturalCD 28 ligands, e.g., a natural ligand for CD28 (e.g., a member of the B7family of proteins, such as B7-1(CD80) and B7-2 (CD86); and anti-CD28monoclonal antibody or fragment thereof capable of crosslinking the CD28molecule, e.g., monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8,248.23.2, and EX5.3D10.

In one embodiment, the molecule providing the primary stimulationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex or CD2, and the costimulatory molecule are coupled tothe same surface.

In certain embodiments, binding agents that provide stimulatory andcostimulatory signals are localized on the surface of a cell. This canbe accomplished by transfecting or transducing a cell with a nucleicacid encoding the binding agent in a form suitable for its expression onthe cell surface or alternatively by coupling a binding agent to thecell surface.

In another embodiment, the molecule providing the primary stimulationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex or CD2, and the costimulatory molecule are displayed onantigen presenting cells.

In one embodiment, the molecule providing the primary stimulationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex or CD2, and the costimulatory molecule are provided onseparate surfaces.

In a certain embodiment, one of the binding agents that providestimulatory and costimulatory signals is soluble (provided in solution)and the other agent(s) is provided on one or more surfaces.

In a particular embodiment, the binding agents that provide stimulatoryand costimulatory signals are both provided in a soluble form (providedin solution).

In various embodiments, the methods for manufacturing T cellscontemplated herein comprise activating T cells with anti-CD3 andanti-CD28 antibodies.

T cell compositions manufactured by the methods contemplated hereincomprise T cells activated and/or expanded in the presence of one ormore agents that inhibit a PI3K cell signaling pathway. T cells modifiedto express an anti-STn CAR can be activated and expanded before and/orafter the T cells are modified. In particular embodiments, a populationof T cells is activated, modified to express an anti-STn CAR, and thencultured for expansion.

In one embodiment, T cells manufactured by the methods contemplatedherein comprise an increased number of T cells expressing markersindicative of high proliferative potential and the ability to self-renewbut that do not express or express substantially undetectable markers ofT cell differentiation. These T cells may be repeatedly activated andexpanded in a robust fashion and thereby provide an improved therapeuticT cell composition.

In one embodiment, a population of T cells activated and expanded in thepresence of one or more agents that inhibit a PI3K cell signalingpathway is expanded at least 1.5 fold, at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 25 fold, atleast 50 fold, at least 100 fold, at least 250 fold, at least 500 fold,at least 1000 fold, or more compared to a population of T cellsactivated and expanded without a PI3K inhibitor.

In one embodiment, a population of T cells characterized by theexpression of markers young T cells are activated and expanded in thepresence of one or more agents that inhibit a PI3K cell signalingpathway is expanded at least 1.5 fold, at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 25 fold, atleast 50 fold, at least 100 fold, at least 250 fold, at least 500 fold,at least 1000 fold, or more compared the population of T cells activatedand expanded without a PI3K inhibitor.

In one embodiment, expanding T cells activated by the methodscontemplated herein further comprises culturing a population of cellscomprising T cells for several hours (about 3 hours) to about 7 days toabout 28 days or any hourly integer value in between. In anotherembodiment, the T cell composition may be cultured for 14 days. In aparticular embodiment, T cells are cultured for about 21 days. Inanother embodiment, the T cell compositions are cultured for about 2-3days. Several cycles of stimulation/activation/expansion may also bedesired such that culture time of T cells can be 60 days or more.

In particular embodiments, conditions appropriate for T cell cultureinclude an appropriate media (e.g., Minimal Essential Media or RPMIMedia 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary forproliferation and viability including, but not limited to serum (e.g.,fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ,IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or anyother additives suitable for the growth of cells known to the skilledartisan.

Further illustrative examples of cell culture media include, but are notlimited to RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 5,and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.

Illustrative examples of other additives for T cell expansion include,but are not limited to, surfactant, piasmanate, pH buffers such asHEPES, and reducing agents such as N-acetyl-cysteine and2-mercaptoethanol

Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% C02).

3. Agents

In various embodiments, a method for manufacturing T cells is providedthat expands undifferentiated or developmentally potent T cellscomprising contacting T cells with an agent that modulates a PI3Kpathway in the cells. In various embodiments, a method for manufacturingT cells is provided that expands undifferentiated or developmentallypotent T cells comprising contacting T cells with an agent thatmodulates a PI3K/AKT/mTOR pathway in the cells. The cells may becontacted prior to, during, and/or after activation and expansion. The Tcell compositions retain sufficient T cell potency such that they mayundergo multiple rounds of expansion without a substantial increase indifferentiation.

As used herein, the terms “modulate,” “modulator,” or “modulatory agent”or comparable term refer to an agent's ability to elicit a change in acell signaling pathway. A modulator may increase or decrease an amount,activity of a pathway component or increase or decrease a desired effector output of a cell signaling pathway. In one embodiment, the modulatoris an inhibitor. In another embodiment, the modulator is an activator.

An “agent” refers to a compound, small molecule, e.g., small organicmolecule, nucleic acid, polypeptide, or a fragment, isoform, variant,analog, or derivative thereof used in the modulation of a PI3K/AKT/mTORpathway.

A “small molecule” refers to a composition that has a molecular weightof less than about 5 kD, less than about 4 kD, less than about 3 kD,less than about 2 kD, less than about 1 kD, or less than about 0.5 kD.Small molecules may comprise nucleic acids, peptides, polypeptides,peptidomimetics, peptoids, carbohydrates, lipids, components thereof orother organic or inorganic molecules. Libraries of chemical and/orbiological mixtures, such as fungal, bacterial, or algal extracts, areknown in the art and can be screened with any of the assays. Examples ofmethods for the synthesis of molecular libraries can be found in:(Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt etal., 1993; Gallop et al., 1994; Zuckermann et al., 1994).

An “analog” refers to a small organic compound, a nucleotide, a protein,or a polypeptide that possesses similar or identical activity orfunction(s) as the compound, nucleotide, protein or polypeptide orcompound having the desired activity, but need not necessarily comprisea sequence or structure that is similar or identical to the sequence orstructure of the preferred embodiment.

A “derivative” refers to either a compound, a protein or polypeptidethat comprises an amino acid sequence of a parent protein or polypeptidethat has been altered by the introduction of amino acid residuesubstitutions, deletions or additions, or a nucleic acid or nucleotidethat has been modified by either introduction of nucleotidesubstitutions or deletions, additions or mutations. The derivativenucleic acid, nucleotide, protein or polypeptide possesses a similar oridentical function as the parent polypeptide.

In various embodiments, the agent that modulates a PI3K pathwayactivates a component of the pathway. An “activator,” or “agonist”refers to an agent that promotes, increases, or induces one or moreactivities of a molecule in a PI3K/AKT/mTOR pathway including, withoutlimitation, a molecule that activates one or more activities of a PI3K.

In various embodiments, the agent that modulates a PI3K pathway inhibitsa component of the pathway. An “inhibitor” or “antagonist” refers to anagent that inhibits, decreases, or reduces one or more activities of amolecule in a PI3K/AKT/mTOR pathway including, without limitation, amolecule than inhibits one or more activities of a PI3K. In oneembodiment, the inhibitor is a dual molecule inhibitor. In particularembodiment, the inhibitor may inhibit a class of molecules have the sameor substantially similar activities (a pan-inhibitor) or mayspecifically inhibit a molecule's activity (a selective or specificinhibitor). Inhibition may also be irreversible or reversible.

In one embodiment, the inhibitor has an IC50 of at least 1 nM, at least2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, atleast 200 nM, at least 500 nM, at least 1 μM, at least 10 μM, at least50 μM, or at least 100 μM. IC50 determinations can be accomplished usingany conventional techniques known in the art. For example, an IC50 canbe determined by measuring the activity of a given enzyme in thepresence of a range of concentrations of the inhibitor under study. Theexperimentally obtained values of enzyme activity then are plottedagainst the inhibitor concentrations used. The concentration of theinhibitor that shows 50% enzyme activity (as compared to the activity inthe absence of any inhibitor) is taken as the “IC50” value. Analogously,other inhibitory concentrations can be defined through appropriatedeterminations of activity.

In various embodiments, T cells are contacted or treated or culturedwith one or more modulators of a PI3K/AKT/mTOR pathway at aconcentration of at least 1 nM, at least 2 nM, at least 5 nM, at least10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500nM, at least 1 μM, at least 10 μM, at least 50 μM, at least 100 μM, orat least 1 M.

In particular embodiments, T cells may be contacted or treated orcultured with one or more modulators of a PI3K/AKT/mTOR pathway for atleast 12 hours, 18 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more rounds of expansion.

The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycinpathway serves as a conduit to integrate growth factor signaling withcellular proliferation, differentiation, metabolism, and survival. PI3Ksare a family of highly conserved intracellular lipid kinases. Class IAPI3Ks are activated by growth factor receptor tyrosine kinases (RTKs),either directly or through interaction with the insulin receptorsubstrate family of adaptor molecules. This activity results in theproduction of phosphatidyl-inositol-3,4,5-trisphospate (PIP3) aregulator of the serine/threonine kinase Akt. mTOR acts through thecanonical PI3K pathway via 2 distinct complexes, each characterized bydifferent binding partners that confer distinct activities. mTORC1 (mTORin complex with PRAS40, raptor, and mLST8/GbL) acts as a downstreameffector of PI3K/Akt signaling, linking growth factor signals withprotein translation, cell growth, proliferation, and survival. mTORC2(mTOR in complex with rictor, mSIN1, protor, and mLST8) acts as anupstream activator of Akt.

Upon growth factor receptor-mediated activation of PI3K, Akt isrecruited to the membrane through the interaction of its pleckstrinhomology domain with PIP3, thus exposing its activation loop andenabling phosphorylation at threonine 308 (Thr308) by the constitutivelyactive phosphoinositide-dependent protein kinase 1 (PDK1). For maximalactivation, Akt is also phosphorylated by mTORC2, at serine 473 (Ser473)of its C-terminal hydrophobic motif. DNA-PK and HSP have also been shownto be important in the regulation of Akt activity. Akt activates mTORC1through inhibitory phosphorylation of TSC2, which along with TSC1,negatively regulates mTORC1 by inhibiting the Rheb GTPase, a positiveregulator of mTORC1. mTORC1 has 2 well-defined substrates, p70S6K(referred to hereafter as S6K1) and 4E-BP1, both of which criticallyregulate protein synthesis. Thus, mTORC1 is an important downstreameffector of PI3K, linking growth factor signaling with proteintranslation and cellular proliferation.

a. PI3K Inhibitors

As used herein, the term “PI3K inhibitor” refers to a nucleic acid,peptide, compound, or small organic molecule that binds to and inhibitsat least one activity of PI3K. The PI3K proteins can be divided intothree classes, class 1 PI3Ks, class 2 PI3Ks, and class 3 PI3Ks. Class 1PI3Ks exist as heterodimers consisting of one of four p110 catalyticsubunits (p110α, p110β, p110δ, and p110γ) and one of two families ofregulatory subunits. In particular embodiments, a PI3K inhibitorpreferably targets the class 1 PI3K inhibitors. In one embodiment, aPI3K inhibitor will display selectivity for one or more isoforms of theclass 1 PI3K inhibitors (i.e., selectivity for p110α, p110β, p110δ, andp110γ or one or more of p110α, p110β, p110δ, and p110γ). In anotherembodiment, a PI3K inhibitor will not display isoform selectivity and beconsidered a “pan-PI3K inhibitor.” In one embodiment, a PI3K inhibitorwill compete for binding with ATP to the PI3K catalytic domain.

In certain embodiments, a PI3K inhibitor can, for example, target PI3Kas well as additional proteins in the PI3K-AKT-mTOR pathway. Inparticular embodiments, a PI3K inhibitor that targets both mTOR and PI3Kcan be referred to as either an mTOR inhibitor or a PI3K inhibitor. API3K inhibitor that only targets PI3K can be referred to as a selectivePI3K inhibitor. In one embodiment, a selective PI3K inhibitor can beunderstood to refer to an agent that exhibits a 50% inhibitoryconcentration with respect to PI3K that is at least 10-fold, at least20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least1000-fold, or more, lower than the inhibitor's IC50 with respect to mTORand/or other proteins in the pathway.

In a particular embodiment, exemplary PI3K inhibitors inhibit PI3K withan IC50 (concentration that inhibits 50% of the activity) of about 200nM or less, preferably about 100 nm or less, even more preferably about60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM,50 μM, 25 μM, 10 μM, 1 μM, or less. In one embodiment, a PI3K inhibitorinhibits PI3K with an IC50 from about 2 nM to about 100 nm, morepreferably from about 2 nM to about 50 nM, even more preferably fromabout 2 nM to about 15 nM.

Illustrative examples of PI3K inhibitors suitable for use in the T cellmanufacturing methods contemplated herein include, but are not limitedto, BKM120 (class 1 PI3K inhibitor, Novartis), XL147 (class 1 PI3Kinhibitor, Exelixis), (pan-PI3K inhibitor, GlaxoSmithKline), and PX-866(class 1 PI3K inhibitor; p110α, p110β, and p110γ isoforms, Oncothyreon).

Other illustrative examples of selective PI3K inhibitors include, butare not limited to BYL719, GSK2636771, TGX-221, AS25242, CAL-101,ZSTK474, and IPI-145.

Further illustrative examples of pan-PI3K inhibitors include, but arenot limited to BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.

In a preferred embodiment, the PI3K inhibitor is ZSTK474.

b. AKT Inhibitors

As used herein, the term “AKT inhibitor” refers to a nucleic acid,peptide, compound, or small organic molecule that inhibits at least oneactivity of AKT. AKT inhibitors can be grouped into several classes,including lipid-based inhibitors (e.g., inhibitors that target thepleckstrin homology domain of AKT which prevents AKT from localizing toplasma membranes), ATP-competitive inhibitors, and allostericinhibitors. In one embodiment, AKT inhibitors act by binding to the AKTcatalytic site. In a particular embodiment, Akt inhibitors act byinhibiting phosphorylation of downstream AKT targets such as mTOR. Inanother embodiment, AKT activity is inhibited by inhibiting the inputsignals to activate Akt by inhibiting, for example, DNA-PK activation ofAKT, PDK-1 activation of AKT, and/or mTORC2 activation of Akt.

AKT inhibitors can target all three AKT isoforms, AKT1, AKT2, AKT3 ormay be isoform selective and target only one or two of the AKT isoforms.In one embodiment, an AKT inhibitor can target AKT as well as additionalproteins in the PI3K-AKT-mTOR pathway. An AKT inhibitor that onlytargets AKT can be referred to as a selective AKT inhibitor. In oneembodiment, a selective AKT inhibitor can be understood to refer to anagent that exhibits a 50% inhibitory concentration with respect to AKTthat is at least 10-fold, at least 20-fold, at least 30-fold, at least50-fold, at least 100-fold, at least 1000-fold, or more lower than theinhibitor's IC50 with respect to other proteins in the pathway.

In a particular embodiment, exemplary AKT inhibitors inhibit AKT with anIC50 (concentration that inhibits 50% of the activity) of about 200 nMor less, preferably about 100 nm or less, even more preferably about 60nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50μM, 25 μM, 10 μM, 1 μM, or less. In one embodiment, an AKT inhibits AKTwith an IC50 from about 2 nM to about 100 nm, more preferably from about2 nM to about 50 nM, even more preferably from about 2 nM to about 15nM.

Illustrative examples of AKT inhibitors for use in combination withauristatin based antibody-drug conjugates include, for example,perifosine (Keryx), MK2206 (Merck), VQD-002 (VioQuest), XL418(Exelixis), GSK690693, GDC-0068, and PX316 (PROLX Pharmaceuticals).

An illustrative, non-limiting example of a selective Akt1 inhibitor isA-674563.

An illustrative, non-limiting example of a selective Akt2 inhibitor isCCT128930.

In particular embodiments, the Akt inhibitor DNA-PK activation of Akt,PDK-1 activation of Akt, mTORC2 activation of Akt, or HSP activation ofAkt.

Illustrative examples of DNA-PK inhibitors include, but are not limitedto, NU7441, PI-103, NU7026, PIK-75, and PP-121.

c. mTOR Inhibitors

The terms “mTOR inhibitor” or “agent that inhibits mTOR” refers to anucleic acid, peptide, compound, or small organic molecule that inhibitsat least one activity of an mTOR protein, such as, for example, theserine/threonine protein kinase activity on at least one of itssubstrates (e.g., p70S6 kinase 1, 4E-BP1, AKT/PKB and eEF2). mTORinhibitors are able to bind directly to and inhibit mTORC1, mTORC2 orboth mTORC1 and mTORC2.

Inhibition of mTORC1 and/or mTORC2 activity can be determined by areduction in signal transduction of the PI3K/Akt/mTOR pathway. A widevariety of readouts can be utilized to establish a reduction of theoutput of such signaling pathway. Some non-limiting exemplary readoutsinclude (1) a decrease in phosphorylation of Akt at residues, includingbut not limited to 5473 and T308; (2) a decrease in activation of Akt asevidenced, for example, by a reduction of phosphorylation of Aktsubstrates including but not limited to Fox01/O3a T24/32, GSK3a/β;S21/9, and TSC2 T1462; (3) a decrease in phosphorylation of signalingmolecules downstream of mTOR, including but not limited to ribosomal S6S240/244, 70S6K T389, and 4EBP1 T37/46; and (4) inhibition ofproliferation of cancerous cells.

In one embodiment, the mTOR inhibitors are active site inhibitors. Theseare mTOR inhibitors that bind to the ATP binding site (also referred toas ATP binding pocket) of mTOR and inhibit the catalytic activity ofboth mTORC1 and mTORC2. One class of active site inhibitors suitable foruse in the T cell manufacturing methods contemplated herein are dualspecificity inhibitors that target and directly inhibit both PI3K andmTOR. Dual specificity inhibitors bind to both the ATP binding site ofmTOR and PI3K. Illustrative examples of such inhibitors include, but arenot limited to: imidazoquinazolines, wortmannin, LY294002, PI-103(Cayman Chemical), SF1126 (Semafore), BGT226 (Novartis), XL765(Exelixis) and NVP-BEZ235 (Novartis).

Another class of mTOR active site inhibitors suitable for use in themethods contemplated herein selectively inhibit mTORC1 and mTORC2activity relative to one or more type I phophatidylinositol 3-kinases,e.g., PI3 kinase α, β, γ, or δ. These active site inhibitors bind to theactive site of mTOR but not PI3K. Illustrative examples of suchinhibitors include, but are not limited to: pyrazolopyrimidines, Torin1(Guertin and Sabatini), PP242(2-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol),PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth), andAZD8055 (Liu et al., Nature Review, 8, 627-644, 2009). I

In one embodiment, a selective mTOR inhibitor refers to an agent thatexhibits a 50% inhibitory concentration (IC50) with respect to mTORC1and/or mTORC2, that is at least 10-fold, at least 20-fold, at least50-fold, at least 100-fold, at least 1000-fold, or more, lower than theinhibitor's IC50 with respect to one, two, three, or more type IPI3-kinases or to all of the type I PI3-kinases.

Another class of mTOR inhibitors for use in particular embodiments arereferred to herein as “rapalogs”. As used herein the term “rapalogs”refers to compounds that specifically bind to the mTOR FRB domain (FKBPrapamycin binding domain), are structurally related to rapamycin, andretain the mTOR inhibiting properties. The term rapalogs excludesrapamycin. Rapalogs include esters, ethers, oximes, hydrazones, andhydroxylamines of rapamycin, as well as compounds in which functionalgroups on the rapamycin core structure have been modified, for example,by reduction or oxidation. Pharmaceutically acceptable salts of suchcompounds are also considered to be rapamycin derivatives. Illustrativeexamples of rapalogs suitable for use in the methods contemplated hereininclude, without limitation, temsirolimus (CC1779), everolimus (RAD001),deforolimus (AP23573), AZD8055 (AstraZeneca), and OSI-027 (OSI).

In one embodiment, the agent is the mTOR inhibitor rapamycin(sirolimus).

In a particular embodiment, exemplary mTOR inhibitors inhibit eithermTORC1, mTORC2 or both mTORC1 and mTORC2 with an IC50 (concentrationthat inhibits 50% of the activity) of about 200 nM or less, preferablyabout 100 nm or less, even more preferably about 60 nM or less, about 25nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1μM, or less. In one embodiment, a mTOR inhibitor inhibits either mTORC1,mTORC2 or both mTORC1 and mTORC2 with an IC50 from about 2 nM to about100 nm, more preferably from about 2 nM to about 50 nM, even morepreferably from about 2 nM to about 15 nM.

In one embodiment, exemplary mTOR inhibitors inhibit either PI3K andmTORC1 or mTORC2 or both mTORC1 and mTORC2 and PI3K with an IC50(concentration that inhibits 50% of the activity) of about 200 nM orless, preferably about 100 nm or less, even more preferably about 60 nMor less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50μM, 25 μM, 10 μM, 1 μM, or less. In one embodiment, a mTOR inhibitorinhibits PI3K and mTORC1 or mTORC2 or both mTORC1 and mTORC2 and PI3Kwith an IC50 from about 2 nM to about 100 nm, more preferably from about2 nM to about 50 nM, even more preferably from about 2 nM to about 15nM.

Further illustrative examples of mTOR inhibitors suitable for use inparticular embodiments include, but are not limited to AZD8055, INK128,rapamycin, PF-04691502, and everolimus.

mTOR has been shown to demonstrate a robust and specific catalyticactivity toward the physiological substrate proteins, p70 S6 ribosomalprotein kinase I (p70S6K1) and eIF4E binding protein 1 (4EBP1) asmeasured by phosphor-specific antibodies in Western blotting.

In one embodiment, the inhibitor of the PI3K/AKT/mTOR pathway is a s6kinase inhibitor selected from the group consisting of: BI-D1870, H89,PF-4708671, FMK, and AT7867.

I. Compositions and Formulations

The compositions contemplated herein may comprise one or morepolypeptides, polynucleotides, vectors comprising same, geneticallymodified immune effector cells, etc., as contemplated herein.Compositions include, but are not limited to pharmaceuticalcompositions. A “pharmaceutical composition” refers to a compositionformulated in pharmaceutically-acceptable or physiologically-acceptablesolutions for administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy. It will alsobe understood that, if desired, the compositions may be administered incombination with other agents as well, such as, e.g., cytokines, growthfactors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs,antibodies, or other various pharmaceutically-active agents. There isvirtually no limit to other components that may also be included in thecompositions, provided that the additional agents do not adverselyaffect the ability of the composition to deliver the intended therapy.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein “pharmaceutically acceptable carrier, diluent orexcipient” includes without limitation any adjuvant, carrier, excipient,glidant, sweetening agent, diluent, preservative, dye/colorant, flavorenhancer, surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, surfactant, or emulsifier which hasbeen approved by the United States Food and Drug Administration as beingacceptable for use in humans or domestic animals. Exemplarypharmaceutically acceptable carriers include, but are not limited to, tosugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate;tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal andvegetable fats, paraffins, silicones, bentonites, silicic acid, zincoxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and any other compatible substancesemployed in pharmaceutical formulations.

In particular embodiments, compositions comprise an amount ofCAR-expressing immune effector cells contemplated herein. As usedherein, the term “amount” refers to “an amount effective” or “aneffective amount” of a genetically modified therapeutic cell, e.g., Tcell, to achieve a beneficial or desired prophylactic or therapeuticresult, including clinical results.

A “prophylactically effective amount” refers to an amount of agenetically modified therapeutic cell effective to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount is less than the therapeuticallyeffective amount.

A “therapeutically effective amount” of a genetically modifiedtherapeutic cell may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of thestem and progenitor cells to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the virus or transduced therapeuticcells are outweighed by the therapeutically beneficial effects. The term“therapeutically effective amount” includes an amount that is effectiveto “treat” a subject (e.g., a patient). When a therapeutic amount isindicated, the precise amount of the compositions to be administered canbe determined by a physician with consideration of individualdifferences in age, weight, tumor size, extent of infection ormetastasis, and condition of the patient (subject). It can generally bestated that a pharmaceutical composition comprising the T cellsdescribed herein may be administered at a dosage of 10² to 10¹⁰ cells/kgbody weight, preferably 10⁵ to 10⁶ cells/kg body weight, including allinteger values within those ranges. The number of cells will depend uponthe ultimate use for which the composition is intended as will the typeof cells included therein. For uses provided herein, the cells aregenerally in a volume of a liter or less, can be 500 mLs or less, even250 mLs or 100 mLs or less. Hence the density of the desired cells istypically greater than 10⁶ cells/ml and generally is greater than 10⁷cells/ml, generally 10⁸ cells/ml or greater. The clinically relevantnumber of immune cells can be apportioned into multiple infusions thatcumulatively equal or exceed 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or10¹² cells. In some embodiments, particularly since all the infusedcells will be redirected to a particular target antigen, lower numbersof cells, in the range of 10⁶/kilogram (10⁶-10¹¹ per patient) may beadministered. CAR expressing cell compositions may be administeredmultiple times at dosages within these ranges. The cells may beallogeneic, syngeneic, xenogeneic, or autologous to the patientundergoing therapy. If desired, the treatment may also includeadministration of mitogens (e.g., PHA) or lymphokines, cytokines, and/orchemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta,GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein toenhance induction of the immune response.

Generally, compositions comprising the cells activated and expanded asdescribed herein may be utilized in the treatment and prevention ofdiseases that arise in individuals who are immunocompromised. Inparticular, compositions comprising the CAR-modified T cellscontemplated herein are used in the treatment of cancer. In particularembodiments, CAR-modified T cells may be administered either alone, oras a pharmaceutical composition in combination with carriers, diluents,excipients, and/or with other components such as IL-2 or other cytokinesor cell populations. In particular embodiments, pharmaceuticalcompositions comprise an amount of genetically modified T cells, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients.

Pharmaceutical compositions comprising a CAR-expressing immune effectorcell population, such as T cells, may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. In particular embodiments, compositionsare preferably formulated for parenteral administration, e.g.,intravascular (intravenous or intraarterial), intraperitoneal orintramuscular administration.

The liquid pharmaceutical compositions, whether they be solutions,suspensions or other like form, may include one or more of thefollowing: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or diglycerides whichmay serve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. An injectablepharmaceutical composition is preferably sterile.

In one embodiment, the T cell compositions contemplated herein areformulated in a pharmaceutically acceptable cell culture medium. Suchcompositions are suitable for administration to human subjects. Inparticular embodiments, the pharmaceutically acceptable cell culturemedium is a serum free medium.

Serum-free medium has several advantages over serum containing medium,including a simplified and better defined composition, a reduced degreeof contaminants, elimination of a potential source of infectious agents,and lower cost. In various embodiments, the serum-free medium isanimal-free, and may optionally be protein-free. Optionally, the mediummay contain biopharmaceutically acceptable recombinant proteins.“Animal-free” medium refers to medium wherein the components are derivedfrom non-animal sources. Recombinant proteins replace native animalproteins in animal-free medium and the nutrients are obtained fromsynthetic, plant or microbial sources. “Protein-free” medium, incontrast, is defined as substantially free of protein.

Illustrative examples of serum-free media used in particularcompositions includes, but is not limited to QBSF-60 (QualityBiological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

In one preferred embodiment, compositions comprising T cellscontemplated herein are formulated in a solution comprising PlasmaLyteA.

In another preferred embodiment, compositions comprising T cellscontemplated herein are formulated in a solution comprising acryopreservation medium. For example, cryopreservation media withcryopreservation agents may be used to maintain a high cell viabilityoutcome post-thaw. Illustrative examples of cryopreservation media usedin particular compositions includes, but is not limited to, CryoStorCS10, CryoStor CS5, and CryoStor CS2.

In a more preferred embodiment, compositions comprising T cellscontemplated herein are formulated in a solution comprising 50:50PlasmaLyte A to CryoStor CS10.

In a particular embodiment, compositions comprise an effective amount ofCAR-expressing immune effector cells, alone or in combination with oneor more therapeutic agents. Thus, the CAR-expressing immune effectorcell compositions may be administered alone or in combination with otherknown cancer treatments, such as radiation therapy, chemotherapy,transplantation, immunotherapy, hormone therapy, photodynamic therapy,etc. The compositions may also be administered in combination withantibiotics. Such therapeutic agents may be accepted in the art as astandard treatment for a particular disease state as described herein,such as a particular cancer. Exemplary therapeutic agents contemplatedinclude cytokines, growth factors, steroids, NSAIDs, DMARDs,anti-inflammatories, chemotherapeutics, radiotherapeutics, therapeuticantibodies, or other active and ancillary agents.

In certain embodiments, compositions comprising CAR-expressing immuneeffector cells disclosed herein may be administered in conjunction withany number of chemotherapeutic agents. Illustrative examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®., Rhne-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid derivatives such asTargretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on cancers such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

A variety of other therapeutic agents may be used in conjunction withthe compositions described herein. In one embodiment, the compositioncomprising CAR-expressing immune effector cells is administered with ananti-inflammatory agent. Anti-inflammatory agents or drugs include, butare not limited to, steroids and glucocorticoids (includingbetamethasone, budesonide, dexamethasone, hydrocortisone acetate,hydrocortisone, hydrocortisone, methylprednisolone, prednisolone,prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs(NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate,sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide andmycophenolate.

Other exemplary NSAIDs are chosen from the group consisting ofibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX®(rofecoxib) and CELEBREX® (celecoxib), and sialylates. Exemplaryanalgesics are chosen from the group consisting of acetaminophen,oxycodone, tramadol of proporxyphene hydrochloride. Exemplaryglucocorticoids are chosen from the group consisting of cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone, orprednisone. Exemplary biological response modifiers include moleculesdirected against cell surface markers (e.g., CD4, CD5, etc.), cytokineinhibitors, such as the TNF antagonists (e.g., etanercept (ENBREL®),adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitorsand adhesion molecule inhibitors. The biological response modifiersinclude monoclonal antibodies as well as recombinant forms of molecules.Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine,methotrexate, penicillamine, leflunomide, sulfasalazine,hydroxychloroquine, Gold (oral (auranofin) and intramuscular) andminocycline.

Illustrative examples of therapeutic antibodies suitable for combinationwith the CAR modified T cells contemplated herein, include but are notlimited to, bavituximab, bevacizumab (avastin), bivatuzumab,blinatumomab, conatumumab, daratumumab, duligotumab, dacetuzumab,dalotuzumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab,inotuzumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab,ocaratuzumab, ofatumumab, rituximab, siltuximab, teprotumumab, andublituximab.

In certain embodiments, the compositions described herein areadministered in conjunction with a cytokine. By “cytokine” as usedherein is meant a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Examplesof such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-beta;platelet-growth factor; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta; insulin-like growth factor-I and —II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, beta, and -gamma; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-lalpha,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12;IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture, and biologically active equivalents of thenative sequence cytokines.

In particular embodiments, a composition comprises CAR T cellscontemplated herein that are cultured in the presence of a PI3Kinhibitor as disclosed herein and express one or more of the followingmarkers: CD3ζ, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, andHLA-DR can be further isolated by positive or negative selectiontechniques. In one embodiment, a composition comprises a specificsubpopulation of T cells, expressing one or more of the markers selectedfrom the group consisting of i) CD62L, CCR7, CD28, CD27, CD122, CD127,CD197; ii) CD62L, CD127, CD197, CD38; and iii) CD62L, CD27, CD127, andCD8, is further isolated by positive or negative selection techniques.In various embodiments, compositions do not express or do notsubstantially express one or more of the following markers: CD57, CD244,CD160, PD-1, CTLA4, TIM3, and LAG3.

In one embodiment, expression of one or more of the markers selectedfrom the group consisting of CD62L, CD127, CD197, and CD38 is increasedat least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, atleast 9 fold, at least 10 fold, at least 25 fold, or more compared to apopulation of T cells activated and expanded without a PI3K inhibitor.

In one embodiment, expression of one or more of the markers selectedfrom the group consisting of CD62L, CD127, CD27, and CD8 is increased atleast 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, atleast 9 fold, at least 10 fold, at least 25 fold, or more compared to apopulation of T cells activated and expanded without a PI3K inhibitor.

In one embodiment, expression of one or more of the markers selectedfrom the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3, andLAG3 is decreased at least 1.5 fold, at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 25 fold, ormore compared to a population of T cells activated and expanded with aPI3K inhibitor.

J. Targets Cells and Antigens

Genetically modified immune effector cells redirected to a target cell,e.g., cancer cell, and that comprise CARs having a binding domain thatbinds to an STn expressing glycoprotein, e.g., TAG-72, on the targetcells.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues.

As used herein, the term “malignant” refers to a cancer in which a groupof tumor cells display one or more of uncontrolled growth (i.e.,division beyond normal limits), invasion (i.e., intrusion on anddestruction of adjacent tissues), and metastasis (i.e., spread to otherlocations in the body via lymph or blood). As used herein, the term“metastasize” refers to the spread of cancer from one part of the bodyto another. A tumor formed by cells that have spread is called a“metastatic tumor” or a “metastasis.” The metastatic tumor containscells that are like those in the original (primary) tumor.

As used herein, the term “benign” or “non-malignant” refers to tumorsthat may grow larger but do not spread to other parts of the body.Benign tumors are self-limited and typically do not invade ormetastasize.

A “cancer cell” refers to an individual cell of a cancerous growth ortissue. Cancer cells include both solid cancers and liquid cancers. A“tumor” or “tumor cell” refers generally to a swelling or lesion formedby an abnormal growth of cells, which may be benign, pre-malignant, ormalignant. Most cancers form tumors, but liquid cancers, e.g., leukemia,do not necessarily form tumors. For those cancers that form tumors, theterms cancer (cell) and tumor (cell) are used interchangeably. Theamount of a tumor in an individual is the “tumor burden” which can bemeasured as the number, volume, or weight of the tumor.

In one embodiment, the target cell expresses an antigen, e.g., a targetantigen that is not substantially found on the surface of other normal(desired) cells.

In one embodiment, the target cell is a bone cell, osteocyte,osteoblast, adipose cell, chondrocyte, chondroblast, muscle cell,skeletal muscle cell, myoblast, myocyte, smooth muscle cell, bladdercell, bone marrow cell, central nervous system (CNS) cell, peripheralnervous system (PNS) cell, glial cell, astrocyte cell, neuron, pigmentcell, epithelial cell, skin cell, endothelial cell, vascular endothelialcell, breast cell, colon cell, esophagus cell, gastrointestinal cell,stomach cell, colon cell, head cell, neck cell, gum cell, tongue cell,kidney cell, liver cell, lung cell, nasopharynx cell, ovary cell,follicular cell, cervical cell, vaginal cell, uterine cell, pancreaticcell, pancreatic parenchymal cell, pancreatic duct cell, pancreaticislet cell, prostate cell, penile cell, gonadal cell, testis cell,hematopoietic cell, lymphoid cell, or myeloid cell.

In one embodiment, the target cell expresses an STn-expressingglycoprotein. In one embodiment, the target cell is a hematopoieticcell, an esophageal cell, a lung cell, an ovarian cell, a cervix cell, apancreatic cell, a cell of the gall bladder or bile duct, a stomachcell, a colon cell, a breast cell, a goblet cell, an enterocyte, a stemcell, an endothelial cell, an epithelial cell, or any cell that expressa glycoepitope, e.g., ST6GALNAC1.

In certain embodiments, the target cell is part of a lining of the gut(e.g., esophagus, stomach, colon), a breast tissue, a lung tissue, acolon tissue, a pancreatic tissue, a gall bladder or bile duct tissue,an ovarian tissue, a cervix tissue, a bladder tissue, a kidney tissue,or an epithelial tissue.

In a particular embodiment, the target cell is a cancer cell or cancerstem cell that expresses an STn expressing glycoprotein.

In one embodiment, the target cell is solid cancer cell that expressesan STn expressing glycoprotein.

Illustrative examples of cells that can be targeted by the compositionsand methods contemplated in particular embodiments include, but are notlimited to those of the following solid cancers: adrenal cancer,adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma,atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile ductcancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer,bronchial tumors, cardiac tumors, cervical cancer, cholangiocarcinoma,chondrosarcoma, chordoma, colon cancer, colorectal cancer,craniopharyngioma, ductal carcinoma in situ (DCIS) endometrial cancer,ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma,extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer,fallopian tube cancer, fibrous histiosarcoma, fibrosarcoma, gallbladdercancer, gastric cancer, gastrointestinal carcinoid tumors,gastrointestinal stromal tumor (GIST), germ cell tumors, glioma,glioblastoma, head and neck cancer, hemangioblastoma, hepatocellularcancer, hypopharyngeal cancer, intraocular melanoma, kaposi sarcoma,kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer,liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lungcarcinoid tumor, malignant mesothelioma, medullary carcinoma,medulloblastoma, menangioma, melanoma, Merkel cell carcinoma, midlinetract carcinoma, mouth cancer, myxosarcoma, myelodysplastic syndrome,myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer,nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer,oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer,pancreatic cancer, pancreatic islet cell tumors, papillary carcinoma,paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer,pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma,primary peritoneal cancer, prostate cancer, rectal cancer,retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer,rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skincancer, soft tissue sarcoma, squamous cell carcinoma, small cell lungcancer, small intestine cancer, stomach cancer, sweat gland carcinoma,synovioma, testicular cancer, throat cancer, thymus cancer, thyroidcancer, urethral cancer, uterine cancer, uterine sarcoma, vaginalcancer, vascular cancer, vulvar cancer, and Wilms Tumor.

In another embodiment, the cell is a solid cancer cell that expressesSTn-expressing glycoprotein. Exemplary STn expressing solid cancer cellsthat may be prevented, treated, or ameliorated with the compositionsinclude, but are not limited to: esophageal cancer, lung cancer, ovariancancer, cervical cancer, pancreatic cancer, choleangiocarcinoma, gastriccancer, colon cancer, bladder cancer, kidney cancer, and breast cancer.

In a particular embodiment, the target cell is a liquid cancer orhematological cancer cell that expresses STn-expressing glycoprotein.

Illustrative examples of liquid cancers or hematological cancers thatmay be prevented, treated, or ameliorated with the compositionscontemplated in particular embodiments include, but are not limited to:leukemias, lymphomas, and multiple myeloma.

Illustrative examples of cells that can be targeted by anti-STN CARscontemplated in particular embodiments include, but are not limited tothose of the following leukemias: acute lymphocytic leukemia (ALL), Tcell acute lymphoblastic leukemia, acute myeloid leukemia (AML),myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia,hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), andchronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML)and polycythemia vera.

Illustrative examples of cells that can be targeted by the compositionsand methods contemplated in particular embodiments include, but are notlimited to those of the following lymphomas: Hodgkin lymphoma, nodularlymphocyte-predominant Hodgkin lymphoma and Non-Hodgkin lymphoma,including but not limited to B-cell non-Hodgkin lymphomas: Burkittlymphoma, small lymphocytic lymphoma (SLL), diffuse large B-celllymphoma, follicular lymphoma, immunoblastic large cell lymphoma,precursor B-lymphoblastic lymphoma, and mantle cell lymphoma; and T-cellnon-Hodgkin lymphomas: mycosis fungoides, anaplastic large celllymphoma, Sézary syndrome, and precursor T-lymphoblastic lymphoma.

Illustrative examples of cells that can be targeted by the compositionsand methods contemplated in particular embodiments include, but are notlimited to those of the following multiple myelomas: overt multiplemyeloma, smoldering multiple myeloma, plasma cell leukemia,non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitaryplasmacytoma of bone, and extramedullary plasmacytoma.

In a particular embodiment, the target cell is a cancer cell or cancerstem cell that expresses STn-expressing glycoprotein.

In another particular embodiment, the target cell is a cancer cell, suchas a cell in a patient with cancer.

K. Therapeutic Methods

The genetically modified immune effector cells contemplated hereinprovide improved methods of adoptive immunotherapy for use in theprevention, treatment, and amelioration cancers that express STnexpressing glycoprotein, e.g., TAG-72, or for preventing, treating, orameliorating at least one symptom associated with an STn expressingcancer.

In various embodiments, the genetically modified immune effector cellscontemplated herein provide improved methods of adoptive immunotherapyfor use in increasing the cytotoxicity in cancer cells that express STnexpressing glycoprotein in a subject or for use in decreasing the numberof cancer cells expressing STn in a subject.

In particular embodiments, the specificity of a primary immune effectorcell is redirected to cells expressing STn expressing glycoproteins,e.g., cancer cells, by genetically modifying the primary immune effectorcell with a CAR contemplated herein. In various embodiments, a viralvector is used to genetically modify an immune effector cell with aparticular polynucleotide encoding a CAR comprising an anti-STn antigenbinding domain that binds STn expressed on a glycoprotein; a hingedomain; a transmembrane (TM) domain, a short oligo- or polypeptidelinker, that links the TM domain to the intracellular signaling domainof the CAR; and one or more intracellular co-stimulatory signalingdomains; and a primary signaling domain.

In one embodiment, a type of cellular therapy where T cells aregenetically modified to express a CAR that targets STn expressingglycoprotein expressed cancer cells, and the CART cell is infused to arecipient in need thereof is provided. The infused cell is able to killdisease causing cells in the recipient. Unlike antibody therapies, CAR Tcells are able to replicate in vivo resulting in long-term persistencethat can lead to sustained cancer therapy.

In one embodiment, the CAR T cells can undergo robust in vivo T cellexpansion and can persist for an extended amount of time. In anotherembodiment, the CAR T cells evolve into specific memory T cells that canbe reactivated to inhibit any additional tumor formation or growth.

In particular embodiments, compositions comprising immune effector cellscomprising the CARs contemplated herein are used in the treatment ofconditions associated with cancer cells or cancer stem cells thatexpress STn expressing glycoprotein.

Illustrative examples of conditions that can be treated, prevented orameliorated using the immune effector cells comprising the CARscontemplated herein include, but are not limited to: a hematologicalcancer, esophageal cancer, lung cancer, ovarian cancer, cervical cancer,pancreatic cancer, choleangiocarcinoma, gastric cancer, colon cancer,bladder cancer, kidney cancer, and breast cancer.

In particular embodiments, compositions comprising CAR-modified T cellscontemplated herein are used in the treatment of solid cancers. Incertain embodiments, the solid cancer is selected from the groupconsisting of: esophageal cancer, lung cancer, ovarian cancer, cervicalcancer, pancreatic cancer, choleangiocarcinoma, gastric cancer, bladdercancer, colon cancer, kidney cancer, and breast cancer.

In a particular embodiment, compositions comprising CAR-modified T cellscontemplated herein are used in the treatment of liquid or hematologicalcancers.

In certain embodiments, the liquid or hematological cancer is selectedfrom the group consisting of: leukemias, lymphomas, and multiplemyelomas.

In certain embodiments, the liquid or hematological cancer is selectedfrom the group consisting of: acute lymphocytic leukemia (ALL), acutemyeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic,monocytic, erythroleukemia, hairy cell leukemia (HCL), chroniclymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronicmyelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma,nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, smalllymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicularlymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosisfungoides, anaplastic large cell lymphoma, Sézary syndrome, precursorT-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma,smoldering multiple myeloma, plasma cell leukemia, non-secretorymyeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma ofbone, and extramedullary plasmacytoma.

In certain embodiments, the liquid or hematological cancer is selectedfrom the group consisting of: acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma(MM), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

In certain embodiments, the liquid or hematological cancer is CLL.

In particular embodiments, methods comprising administering atherapeutically effective amount of CAR-expressing immune effector cellscontemplated herein or a composition comprising the same, to a patientin need thereof, alone or in combination with one or more therapeuticagents, are provided. In certain embodiments, the cells are used in thetreatment of patients at risk for developing a condition associated withcancer cells that express STn expressing glycoprotein. Thus, inparticular embodiments, methods for the treatment or prevention oramelioration of at least one symptom of cancer comprising administeringto a subject in need thereof, a therapeutically effective amount of theCAR-modified cells contemplated herein.

As used herein, the terms “individual” and “subject” are often usedinterchangeably and refer to any animal that exhibits a symptom of adisease, disorder, or condition that can be treated with the genetherapy vectors, cell-based therapeutics, and methods contemplatedelsewhere herein. In preferred embodiments, a subject includes anyanimal that exhibits symptoms of a disease, disorder, or conditionrelated to cancer that can be treated with the gene therapy vectors,cell-based therapeutics, and methods contemplated elsewhere herein.Suitable subjects (e.g., patients) include laboratory animals (such asmouse, rat, rabbit, or guinea pig), farm animals, and domestic animalsor pets (such as a cat or dog). Non-human primates and, preferably,human patients, are included. Typical subjects include human patientsthat have, have been diagnosed with, or are at risk or having a cancerthat expresses STn on a glycoprotein.

As used herein, the term “patient” refers to a subject that has beendiagnosed with a particular disease, disorder, or condition that can betreated with the gene therapy vectors, cell-based therapeutics, andmethods disclosed elsewhere herein.

As used herein “treatment” or “treating,” includes any beneficial ordesirable effect on the symptoms or pathology of a disease orpathological condition, and may include even minimal reductions in oneor more measurable markers of the disease or condition being treated.Treatment can involve optionally either the reduction of the disease orcondition, or the delaying of the progression of the disease orcondition, e.g., delaying tumor outgrowth. “Treatment” does notnecessarily indicate complete eradication or cure of the disease orcondition, or associated symptoms thereof.

As used herein, “prevent,” and similar words such as “prevented,”“preventing” etc., indicate an approach for preventing, inhibiting, orreducing the likelihood of the occurrence or recurrence of, a disease orcondition. It also refers to delaying the onset or recurrence of adisease or condition or delaying the occurrence or recurrence of thesymptoms of a disease or condition. As used herein, “prevention” andsimilar words also includes reducing the intensity, effect, symptomsand/or burden of a disease or condition prior to onset or recurrence ofthe disease or condition.

As used herein, the phrase “ameliorating at least one symptom of” refersto decreasing one or more symptoms of the disease or condition for whichthe subject is being treated. In particular embodiments, the disease orcondition being treated is a cancer, wherein the one or more symptomsameliorated include, but are not limited to, weakness, fatigue,shortness of breath, easy bruising and bleeding, frequent infections,enlarged lymph nodes, distended or painful abdomen (due to enlargedabdominal organs), bone or joint pain, fractures, unplanned weight loss,poor appetite, night sweats, persistent mild fever, and decreasedurination (due to impaired kidney function).

By “enhance” or “promote,” or “increase” or “expand” refers generally tothe ability of a composition contemplated herein, e.g., a geneticallymodified T cell or vector encoding a CAR, to produce, elicit, or cause agreater physiological response (i.e., downstream effects) compared tothe response caused by either vehicle or a control molecule/composition.A measurable physiological response may include an increase in T cellexpansion, activation, persistence, and/or an increase in cancer cellkilling ability, among others apparent from the understanding in the artand the description herein. An “increased” or “enhanced” amount istypically a “statistically significant” amount, and may include anincrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30or more times (e.g., 500, 1000 times) (including all integers anddecimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.)the response produced by vehicle or a control composition.

By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refersgenerally to the ability of composition contemplated herein to produce,elicit, or cause a lesser physiological response (i.e., downstreameffects) compared to the response caused by either vehicle or a controlmolecule/composition. A “decrease” or “reduced” amount is typically a“statistically significant” amount, and may include an decrease that is1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response(reference response) produced by vehicle, a control composition, or theresponse in a particular cell lineage.

By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “nosubstantial change,” or “no substantial decrease” refers generally tothe ability of a composition contemplated herein to produce, elicit, orcause a substantially similar or comparable physiological response(i.e., downstream effects) in a cell, as compared to the response causedby either vehicle, a control molecule/composition, or the response in aparticular cell lineage. A comparable response is one that is notsignificantly different or measurable different from the referenceresponse.

In one embodiment, a method of treating cancer in a subject in needthereof comprises administering an effective amount, e.g.,therapeutically effective amount of a composition comprising geneticallymodified immune effector cells contemplated herein. The quantity andfrequency of administration will be determined by such factors as thecondition of the patient, and the type and severity of the patient'sdisease, although appropriate dosages may be determined by clinicaltrials.

In one embodiment, the amount of immune effector cells, e.g., T cells,in the composition administered to a subject is at least 0.1×10⁵ cells,at least 0.5×10⁵ cells, at least 1×10⁵ cells, at least 5×10⁵ cells, atleast 1×10⁶ cells, at least 0.5×10⁷ cells, at least 1×10⁷ cells, atleast 0.5×10⁸ cells, at least 1×10⁸ cells, at least 0.5×10⁹ cells, atleast 1×10⁹ cells, at least 2×10⁹ cells, at least 3×10⁹ cells, at least4×10⁹ cells, at least 5×10⁹ cells, or at least 1×10¹⁰ cells. Inparticular embodiments, about 1×10⁷ T cells to about 1×10⁹ T cells,about 2×10⁷ T cells to about 0.9×10⁹ T cells, about 3×10⁷ T cells toabout 0.8×10⁹ T cells, about 4×10⁷ T cells to about 0.7×10⁹ T cells,about 5×10⁷ T cells to about 0.6×10⁹ T cells, or about 5×10⁷ T cells toabout 0.5×10⁹ T cells are administered to a subject.

In one embodiment, the amount of immune effector cells, e.g., T cells,in the composition administered to a subject is at least 0.1×10⁴cells/kg of bodyweight, at least 0.5×10⁴ cells/kg of bodyweight, atleast 1×10⁴ cells/kg of bodyweight, at least 5×10⁴ cells/kg ofbodyweight, at least 1×10⁵ cells/kg of bodyweight, at least 0.5×10⁶cells/kg of bodyweight, at least 1×10⁶ cells/kg of bodyweight, at least0.5×10⁷ cells/kg of bodyweight, at least 1×10⁷ cells/kg of bodyweight,at least 0.5×10⁸ cells/kg of bodyweight, at least 1×10⁸ cells/kg ofbodyweight, at least 2×10⁸ cells/kg of bodyweight, at least 3×10⁸cells/kg of bodyweight, at least 4×10⁸ cells/kg of bodyweight, at least5×10⁸ cells/kg of bodyweight, or at least 1×10⁹ cells/kg of bodyweight.In particular embodiments, about 1×10⁶ T cells/kg of bodyweight to about1×10⁸ T cells/kg of bodyweight, about 2×10⁶ T cells/kg of bodyweight toabout 0.9×10⁸ T cells/kg of bodyweight, about 3×10⁶ T cells/kg ofbodyweight to about 0.8×10⁸ T cells/kg of bodyweight, about 4×10⁶ Tcells/kg of bodyweight to about 0.7×10⁸ T cells/kg of bodyweight, about5×10⁶ T cells/kg of bodyweight to about 0.6×10⁸ T cells/kg ofbodyweight, or about 5×10⁶ T cells/kg of bodyweight to about 0.5×10⁸ Tcells/kg of bodyweight are administered to a subject.

One of ordinary skill in the art would recognize that multipleadministrations of the compositions contemplated herein may be requiredto effect the desired therapy. For example a composition may beadministered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a spanof 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.

In certain embodiments, it may be desirable to administer activatedimmune effector cells to a subject and then subsequently redraw blood(or have an apheresis performed), activate immune effector cellstherefrom, and reinfuse the patient with these activated and expandedimmune effector cells. This process can be carried out multiple timesevery few weeks. In certain embodiments, immune effector cells can beactivated from blood draws of from 10 cc to 400 cc. In certainembodiments, immune effector cells are activated from blood draws of 20cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200cc, 250 cc, 300 cc, 350 cc, or 400 cc or more. Not to be bound bytheory, using this multiple blood draw/multiple reinfusion protocol mayserve to select out certain populations of immune effector cells.

The administration of the compositions contemplated herein may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. In apreferred embodiment, compositions are administered parenterally. Thephrases “parenteral administration” and “administered parenterally” asused herein refers to modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravascular, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intratumoral, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion. In one embodiment, the compositions contemplatedherein are administered to a subject by direct injection into a tumor,lymph node, or site of infection.

In one embodiment, a subject in need thereof is administered aneffective amount of a composition to increase a cellular immune responseto a cancer in the subject. The immune response may include cellularimmune responses mediated by cytotoxic T cells capable of killinginfected cells, regulatory T cells, and helper T cell responses. Humoralimmune responses, mediated primarily by helper T cells capable ofactivating B cells thus leading to antibody production, may also beinduced. A variety of techniques may be used for analyzing the type ofimmune responses induced by the compositions, which are well describedin the art; e.g., Current Protocols in Immunology, Edited by: John E.Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, WarrenStrober (2001) John Wiley & Sons, NY, N.Y.

In the case of T cell-mediated killing, CAR-ligand binding initiates CARsignaling to the T cell, resulting in activation of a variety of T cellsignaling pathways that induce the T cell to produce or release proteinscapable of inducing target cell apoptosis by various mechanisms. These Tcell-mediated mechanisms include (but are not limited to) the transferof intracellular cytotoxic granules from the T cell into the targetcell, T cell secretion of pro-inflammatory cytokines that can inducetarget cell killing directly (or indirectly via recruitment of otherkiller effector cells), and up regulation of death receptor ligands(e.g. FasL) on the T cell surface that induce target cell apoptosisfollowing binding to their cognate death receptor (e.g. Fas) on thetarget cell.

In one embodiment, a method of treating a subject diagnosed with acancer that expresses STn on a glycoprotein is provided comprisingremoving immune effector cells from a subject diagnosed with the STnexpressing cancer, genetically modifying said immune effector cells witha vector comprising a nucleic acid encoding a CAR as contemplatedherein, thereby producing a population of modified immune effectorcells, and administering the population of modified immune effectorcells to the same subject. In a preferred embodiment, the immuneeffector cells comprise T cells.

In certain embodiments, methods for stimulating an immune effector cellmediated immune modulator response to a target cell population in asubject are provided comprising the steps of administering to thesubject an immune effector cell population expressing a nucleic acidconstruct encoding a CAR molecule.

The methods for administering the cell compositions contemplated inparticular embodiments includes any method which is effective to resultin reintroduction of ex vivo genetically modified immune effector cellsthat either directly express a CAR in the subject or on reintroductionof the genetically modified progenitors of immune effector cells that onintroduction into a subject differentiate into mature immune effectorcells that express the CAR. One method comprises transducing peripheralblood T cells ex vivo with a nucleic acid construct in accordance withthe contemplated herein and returning the transduced cells into thesubject.

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings contemplated herein that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims. The following examples areprovided by way of illustration only and not by way of limitation. Thoseof skill in the art will readily recognize a variety of noncriticalparameters that could be changed or modified to yield essentiallysimilar results.

EXAMPLES Example 1 Construction of Anti-STn Cars

CARs containing humanized anti-STn scFv antibodies were designed tocontain an MND promoter operably linked to anti-STn scFv, a hinge andtransmembrane domain from CD8α and a CD137 co-stimulatory domainsfollowed by the intracellular signaling domain of the CD3ζ chain.FIG. 1. The anti-STn CARs comprise a CD8α signal peptide (SP) sequencefor the surface expression on immune effector cells. Table 3 shows theIdentity, Genbank Reference, Source Name and Citation for the variousnucleotide segments of an exemplary anti-STn CAR lentiviral vector.

TABLE 3 Nucleotides Identity GenBank Reference Source Name Citation 1-185 pUC19 plasmid Accession #L09137.2 pUC19 New England backbone nt1-185 Biolabs 185-222 Linker Not applicable Synthetic Not applicable223-800 CMV Not Applicable pHCMV (1994) PNAS 91: 9564-68  801-1136 R,U5, PBS, and Accession #M19921.2 pNL4-3 Maldarelli, et. al. packagingsequences nt 454-789 (1991) J Virol: 65(11): 5732-43 1137-1139 Gag startcodon (ATG) Not Applicable Synthetic Not applicable changed to stopcodon (TAG) 1140-1240 HIV-1 gag sequence Accession #M19921.2 pNL4-3Maldarelli, et. al. nt 793-893 (1991) J Virol: 65(11): 5732-43 1241-1243HIV-1 gag sequence Not Applicable Synthetic Not applicable changed to asecond slop codon 1244-1595 HIV-1 gag sequence Accession #M19921.2pNL4-3 Maldarelli, et. al. nt 897-1248 (1991) J Virol: 65(11): 5732-431596-1992 HIV-1 pol Accession #M19921.2 pNL4-3 Maldarelli, et. al.cPPT/CTS nt 4745-5125 (1991) J Virol: 65(11): 5732-43 1993-2517 HIV-1,isolate HXB3 Accession #M14100.1 PgTAT-CMV Malim, M. H. env region (RRE)nt 1875-2399 Nature (1988) 335: 181-183 2518-2693 HIV-1 env sequencesAccession #M19921.2 pNL4-3 Maldarelli, et. al. S/A nt 8290-8470 (1991) JVirol: 65(11): 5732-43 2694-2708 Linker Not applicable Synthetic Notapplicable 2709-3096 MND Not applicable rSPA.mPro.M Challita et al. ND(1995) J. Virol. 69: 748-755 3097-3125 Linker Not applicable SyntheticNot applicable 3126-3188 Signal peptide Synthetic Not applicablevariable Anti-STn scFv Not applicable Synthetic Not applicable 3927-3935Linker Not applicable Synthetic Not applicable 3936-4142 CD8a hinge andTM Accession # Synthetic Milone et al NM_001768 (2009) Mol Ther 17(8):1453-64 4143-4268 CD137 (4-1BB) Accession # Synthetic Milone et alsignaling domain NM_001561 (2009) Mol Ther 17(8): 1453-64 4269-4607CD3-ζ signaling Accession # Synthetic Milone et al domain NM_000734(2009) Mol Ther 17(8): 1453-64 4608-4718 HIV-1 ppt and part Accession#M19921.2 pNL4-3 Maldarelli, et. al. of U3 nt 9005-9110 (1991) J Virol:65(11): 5732-43 4719-4835 HIV-1 part of U3 Accession #M19921.2 pNL4-3Maldarelli, et. al. (399 bp deletion) nt 9511-9627 (1991) and R J Virol:65(11): 5732-43 4836-4859 Synthetic poly(A) Not applicable SyntheticLevitt, N. Genes & Dev (1989) 3: 1019-1025 4860-4878 Linker Notapplicable Synthetic Not Applicable¹ 4879-7351 pUC19 backbone Accession#L09137.2 pUC19 New England nt 2636-2686 Biolabs (Attached)

Example 2 Cytotoxicity of Anti-STn Car T Cells in an In Vitro Model ofHuman Colorectal Cancer

CAR T cells comprising an anti-STn CAR that has a light chain variableregion according to SEQ ID NO: 15 and a heavy chain variable regionaccording to SEQ ID NO: 16 showed selective killing of LS174T cells inan in vitro model of colorectal carcinoma. LS174T cells are a humancolorectal cancer cell line that expresses high levels of STn expressingTAG-72 glycoprotein.

Expression of STn expressing TAG-72 glycoprotein on LS174T cells wasconfirmed by immunohistochemistry. 5×10⁵ LS174T cells were suspended inPBS 100 μL containing 1% bovine serum albumin (BSA), and 5 μg of STnspecific antibody (3E8). The reaction was incubated for 30 minutes at 4°C., then the cells were washed twice with PBS containing 1% BSA. Then, 1μL of mouse anti-human IgG-PE (Southern Biotech) was added and themixture was incubated for 30 minutes at 4° C. Then, the cells werewashed twice with PBS containing 1% BSA. STn expressing TAG-72glycoprotein expression was confirmed with flow cytometry. 98% of LS174Tcells expressed STn expressing TAG-72 glycoprotein. 95% of cells fromthe human stomach cancer cell line also showed STn expression. Incontrast, only about 2% of TAG-72 negative human umbilical veinendotheliocyte cells (HUVEC) cells expressed STn.

Cytotoxic activity of anti-STn CART cells on LS174T cells was assayedusing CellTox™ Green. 1×10⁴ LS174T cells were suspended in 50 μL ofculture medium and combined with 2.0 μL of CellTox™ Green was added. Themixture was aliquoted in a black 96 well plate and aliquots werecombined with either 5×10⁴, 1×10⁵, 2×10⁵, or 3×10⁵ anti-STn CART cellsin 50 μl of culture medium containing human serum and IL-2 (E:T ratio=5,10, 20, 30). After 24 hours of culture at 37° C., the cytotoxicity ascalculated as follows.

CellTox™ Green Fluorescence (RFU)={Reaction of T cells andE-cells}−(Reaction of E-cells)}−(reaction of T cells, background)

The results showed that anti-STn-CAR T cells effectively kill STn-TAG-72expressing LS174T cancer cells compared to STn-absent HUVECs or comparedto control T cells that do not express an anti-STn CAR.

Example 3 Anti-STn Car Expression and Vector Copy Number in Transduced TCells

CAR T cell cultures were established using a system directly scalable tolarge clinical manufacturing processes. Briefly, peripheral bloodmononuclear cells (PBMC) were cultured in static flasks in mediacontaining IL-2 (Cell Genix), antibodies specific for CD3 and CD28(Miltenyi Biotec) and ZSTK474 (Selleckchem). 2×10⁸ transducing units oflentivirus encoding an anti-STn CAR that has a light chain variableregion according to SEQ ID NO: 15 and a heavy chain variable regionaccording to SEQ ID NO: 16 were added one day after culture initiation.The anti-STN CAR T cells were maintained in log-phase by adding freshmedia containing IL-2 and ZSTK474 for a total of ten days of culture. Atthe end of culture, cell surface expression of the anti-STN CAR andvector copy number (VCN) were measured in transduced T cells obtainedfrom three donors.

VCN was assessed by quantitative PCR at the end of culture using primersthat detect the vector or the genomic control RNAseP. The data foranti-STn CAR VCN in the transduced T cells from the three donorscompared to the untransduced T cells are shown in FIG. 3A. Transduced Tcells had a VCN of about 4.5 compared to undetectable VCN in theuntransduced control cells.

Expression of the anti-STn CAR on the surface of T cells was assessed byflow cytometry at the end of culture. Anti-STn CAR T cells werespecifically identified using a two-step detection method ofbiotinylated protein L followed by streptavidin-PE. T cells from threenormal donors showed high expression of the anti-STn CAR. The results ofthis experiment are shown in FIG. 3B.

Example 4 Antigen Specific Cytotoxicity of Anti-STn Car T Cells

Anti-STn CAR T cells were produced as described in Example 3. At the endof culture, the anti-STn CAR T cells were assayed for antigen-specificresponses in two independent assays.

The ability of anti-STn CAR T cells to respond and produce IFNγ to celllines expressing STn on TAG72 was examined using a bead-based cytokineassay (Luminex). Equivalent numbers of tumor cells and anti-STn CAR Tcells (5×10⁴ each) were co-cultured for 24 hours. Supernatants werecollected and the amount of IFNγ produced was quantified by Luminex.Anti-STn CAR T cells produced little to no IFNγ when cultured withouttumor cells. No IFNγ was produced by anti-STn CAR T cells whenco-cultured with tumor cells that do not express STn on their cellsurface in two-dimensional culture (A549 and HDLM-2). When anti-STn CART cells were co-cultured with tumor cells that express STn on TAG72(Jurkat and LS174T), there was a marked increase in amount of IFNγproduced. FIG. 4A.

Cytolysis of LS174T cells co-cultured with anti-STn CAR T cells wasanalyzed. Cytolysis was monitored in real time with an iCELLigenceinstrument capable of monitoring electrical impedance of live cells.Anti-STn CAR T cells, but not untransduced control T cells, inducedcytolysis in co-cultured LS174T cells. FIG. 4B.

Anti-STn CAR T cells (Effector cells) were co-cultured with LS174T cells(Target cells) at different Effector:Target cell ratios. Anti-STn CAR Tcells increase toxicity in a dose dependent manner in co-cultured LS174Tcells. FIG. 4C.

Example 5 Anti-STn Car T Cells Delay Tumor Outgrowth in an Aggressive InVivo Tumor Model

Anti-STn CAR T cells were produced as described in Example 3. Theanti-tumor activity of the CAR T cells was examined in an aggressive invivo tumor model. Animals received a sub cutaneous administration ofcolon adenocarcinoma cells (LS174T) and were infused with equivalent CART cell doses (2×10⁷ T cells) the following day. Control CAR T cells,which comprise truncated CARs that lack the ability to transducesignals, had no effect on tumor growth compared to vehicle-treatedanimals. Anti-STN CAR T cells delayed tumor outgrowth in this aggressivecolon cancer model. FIG. 5.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific embodiments disclosed inthe specification and the claims, but should be construed to include allpossible embodiments along with the full scope of equivalents to whichsuch claims are entitled. Accordingly, the claims are not limited by thedisclosure.

1. A chimeric antigen receptor (CAR) comprising: an extracellular domainthat comprises: a) an anti-sialyl Tn (STn) antibody or antigen bindingfragment thereof that binds one or more epitopes of an STn antigenexpressed on a glycoprotein, wherein the anti-STn antibody or antigenbinding fragment thereof comprises a variable light chain sequencecomprising CDRL1-CDRL3 sequences set forth in SEQ ID NOs: 1-3, 9-11, or17-19, and a variable heavy chain sequence comprising CDRH1-CDRH3sequences set forth in SEQ ID NOs: 4-6, 12-14, or 20-22; b) atransmembrane domain; c) one or more intracellular co-stimulatorysignaling domains; and d) a primary signaling domain.
 2. The CAR ofclaim 1, wherein the STn antigen is expressed on a glycoprotein selectedfrom the group consisting of mucins or mucin-like glycoproteins.
 3. TheCAR of claim 1, wherein the STn antigen is expressed on a mucin selectedfrom the group consisting of: mucin 1 and mucin
 16. 4. The CAR of claim1, wherein the STn antigen is expressed on a mucin-like protein.
 5. TheCAR of claim 1, wherein the STn antigen is expressed on tumor-associatedglycoprotein 72 (TAG-72).
 6. The CAR of claim 1, wherein the anti-STnantibody or antigen binding fragment is selected from the groupconsisting of: a Camel Ig, Ig NAR, Fab fragments, Fab′ fragments,F(ab)′2 fragments, F(ab)′3 fragments, Fv, single chain Fv antibody(“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody,disulfide stabilized Fv protein (“dsFv”), and single-domain antibody(sdAb, Nanobody).
 7. The CAR of claim 1, wherein the anti-STn antibodyor antigen binding fragment is an scFv.
 8. The CAR of claim 1, whereinthe anti-STn antibody or antigen binding fragment thereof comprises oneor more light chain CDRs as set forth in any one of SEQ ID NOs: 1-3and/or one or more heavy chain CDRs as set forth in any one of SEQ IDNOs: 4-6.
 9. The CAR of claim 1, wherein the anti-STn antibody orantigen binding fragment thereof comprises one or more light chain CDRsas set forth in any one of SEQ ID NOs: 9-11 and/or one or more heavychain CDRs as set forth in any one of SEQ ID NOs: 12-14.
 10. The CAR ofclaim 1, wherein the anti-STn antibody or antigen binding fragmentthereof comprises one or more light chain CDRs as set forth in any oneof SEQ ID NOs: 17-19 and/or one or more heavy chain CDRs as set forth inany one of SEQ ID NOs: 20-22.
 11. The CAR of claim 1, wherein theanti-STn antibody or antigen binding fragment thereof comprises avariable light chain sequence as set forth in SEQ ID NO: 7 and/or avariable heavy chain sequence as set forth in SEQ ID NO:
 8. 12. The CARof claim 1, wherein the anti-STn antibody or antigen binding fragmentthereof comprises a variable light chain sequence as set forth in SEQ IDNO: 15 and/or a variable heavy chain sequence as set forth in SEQ ID NO:16.
 13. The CAR of claim 1, wherein the anti-STn antibody or antigenbinding fragment thereof comprises a variable light chain sequence asset forth in SEQ ID NO: 23 and/or a variable heavy chain sequence as setforth in SEQ ID NO:
 24. 14. The CAR of claim 1, wherein thetransmembrane domain is isolated from a polypeptide selected from thegroup consisting of: alpha or beta chain of the T-cell receptor, CDδ,CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33,CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, and PD1. 15.The CAR of claim 1, wherein the transmembrane domain is isolated from apolypeptide selected from the group consisting of: CD8α; CD4, CD45, PD1,and CD152.
 16. The CAR of claim 1, wherein the transmembrane domain isisolated from CD8α. 17.-18. (canceled)
 19. The CAR of claim 1, whereinthe one or more co-stimulatory signaling domains are isolated from aco-stimulatory molecule selected from the group consisting of: TLR1,TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7,CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB),CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.
 20. The CAR ofclaim 1, wherein the one or more co-stimulatory signaling domains areisolated from a co-stimulatory molecule selected from the groupconsisting of: CD28, CD134, and CD137.
 21. The CAR of claim 1, whereinthe one or more co-stimulatory signaling domains is isolated from CD28.22. The CAR of claim 1, wherein the one or more co-stimulatory signalingdomains is isolated from CD134.
 23. The CAR of claim 1, wherein the oneor more co-stimulatory signaling domains is isolated from CD137.
 24. TheCAR of claim 1, wherein the primary signaling domain isolated from apolypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ,CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.
 25. The CAR of claim 1,wherein the primary signaling domain isolated from a CD3.
 26. The CAR ofclaim 1, further comprising a hinge region polypeptide.
 27. The CAR ofclaim 26, wherein the hinge region polypeptide comprises a hinge regionof CD8α.
 28. The CAR of claim 26, wherein the hinge region polypeptidecomprises a hinge region of PD1.
 29. The CAR of claim 26, wherein thehinge region polypeptide comprises a hinge region of CD152.
 30. The CARof claim 1, further comprising a spacer region.
 31. The CAR of claim 30,wherein the spacer region polypeptide comprises CH2 and CH3 regions ofIgG1, IgG4, or IgD.
 32. The CAR of claim 1, further comprising a signalpeptide.
 33. The CAR of claim 32, wherein the signal peptide comprisesan IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or ahuman GM-CSF receptor alpha signal polypeptide.
 34. The CAR of claim 1,wherein the CAR comprises an amino acid sequence set forth in any one ofSEQ ID NOs: 25-27.
 35. A polypeptide comprising the amino acid sequenceof the CAR of claim
 1. 36.-96. (canceled)